Nicholas Dixon

Corrigendum to “Crystal structures and biochemical characterization of DNA sliding clamps from three Gram-negative bacterial pathogens” [J. Struct. Biol. 204 (3) (2018) 396–405]

Publisher: Elsevier BV

Date: 08-2019

DOI: 10.1016/J.JSB.2019.05.003

Replisome speed determines the efficiency of the Tus-Ter replication termination barrier.

Publisher: Springer Science and Business Media LLC

Date: 31-08-2015

DOI: 10.1038/NATURE14866

Abstract: In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites. Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first ex le where intrinsic differences in rates of in idual replisomes have different biological outcomes.

Nitsche’s method for two and three dimensional NURBS patch coupling

Publisher: Springer Science and Business Media LLC

Date: 05-12-2013

DOI: 10.1007/S00466-013-0955-3

Structure and function of a spectrin-like regulator of bacterial cytokinesis

Publisher: Springer Science and Business Media LLC

Date: 18-11-2014

DOI: 10.1038/NCOMMS6421

Abstract: Bacterial cell ision is facilitated by a molecular machine—the isome—that assembles at mid-cell in iding cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the isome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound isome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The in idual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.

Translational incorporation of L‐3,4‐dihydroxyphenylalanine into proteins

Publisher: Wiley

Date: 06-2005

DOI: 10.1111/J.1742-4658.2005.04735.X

Abstract: An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems

Publisher: Proceedings of the National Academy of Sciences

Date: 25-09-2001

DOI: 10.1073/PNAS.191384398

Abstract: The interaction between DNA polymerases and sliding cl proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. The cl proteins also participate in a variety of dynamic and competing protein–protein interactions. However, cl -protein binding sequences have not so far been identified in the eubacteria. Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein–protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL[SD]LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding cl (the β subunit of Escherichia coli DNA polymerase III holoenzyme). Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PolC, PolB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family. The component tripeptide DLF inhibits the binding of the α (DnaE) subunit of E. coli DNA polymerase III to β at μM concentration, identifying key residues. Comparison of the eubacterial, eukaryotic, and archaeal sliding cl binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape.

NMR Spectroscopic Assignment of Backbone and Side-Chain Protons in Fully Protonated Proteins: Microcrystals, Sedimented Assemblies, and Amyloid Fibrils

Publisher: Wiley

Date: 16-11-2016

DOI: 10.1002/ANIE.201607084

Abstract: We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of s le. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis

Publisher: Elsevier BV

Date: 04-2005

DOI: 10.1074/JBC.M414187200

The proofreading exonuclease subunit ε of Escherichia coli DNA polymerase III is tethered to the polymerase subunit α via a flexible linker

Publisher: Oxford University Press (OUP)

Date: 28-07-2008

DOI: 10.1093/NAR/GKN489

Hydrolysis of the 5′-p-nitrophenyl ester of TMP by the proofreading exonuclease (ε) subunit of Escherichia coli DNA polymerase III

Publisher: American Chemical Society (ACS)

Date: 27-03-2002

DOI: 10.1021/BI0159480

Abstract: The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta). The epsilon subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (epsilon 186). A spectrophotometric assay for measurement of epsilon activity is described. Proteins epsilon and epsilon 186 and the epsilon 186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K(M), confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon. On the other hand, formation of the complex with theta stabilized epsilon 186 by approximately 14 degrees C against thermal inactivation. For epsilon 186, k(cat) = 293 min(-)(1) and K(M) = 1.08 mM at pH 8.00 and 25 degrees C, with a Mn(2+) concentration of 1 mM. Hydrolysis of pNP-TMP by epsilon 186 depended absolutely on alent metal ions, and was inhibited by the product TMP. Dependencies on Mn(2+) and Mg(2+) concentrations were examined, giving a K(Mn) of 0.31 mM and a k(cat) of 334 min(-1) for Mn(2+) and a K(Mg) of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg(2+). Inhibition by TMP was formally competitive [K(i) = 4.3 microM (with a Mn(2+) concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by epsilon 186, in the pH range of 6.5-9.0, was found to be simple. K(M) was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) of 7.7, approaching limiting values of 50 min(-1) at pH 9.0. These data are used in conjunction with crystal structures of the complex of epsilon 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by epsilon at high and low pH values.

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase

Publisher: Oxford University Press (OUP)

Date: 20-03-2023

DOI: 10.1093/NAR/GKAD186

Abstract: Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by in idual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging in idual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.

Rational Design of a 3₁₀‐Helical PIP‐Box Mimetic Targeting PCNA, the Human Sliding Clamp

Publisher: Wiley

Date: 19-07-2018

DOI: 10.1002/CHEM.201801734

Abstract: The human sliding cl (PCNA) controls access to DNA for many proteins involved in DNA replication and repair. Proteins are recruited to the PCNA surface by means of a short, conserved peptide motif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as a potentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibitor p21. These mimetics incorporate covalent i,i+4 side-chain/side-chain lactam linkages of different lengths, designed to constrain the peptides into the 3

The E. coli DNA Replication Fork

Publisher: Elsevier

Date: 2016

DOI: 10.1016/BS.ENZ.2016.04.001

Abstract: DNA replication in Escherichia coli initiates at oriC, the origin of replication and proceeds bidirectionally, resulting in two replication forks that travel in opposite directions from the origin. Here, we focus on events at the replication fork. The replication machinery (or replisome), first assembled on both forks at oriC, contains the DnaB helicase for strand separation, and the DNA polymerase III holoenzyme (Pol III HE) for DNA synthesis. DnaB interacts transiently with the DnaG primase for RNA priming on both strands. The Pol III HE is made up of three subassemblies: (i) the αɛθ core polymerase complex that is present in two (or three) copies to simultaneously copy both DNA strands, (ii) the β2 sliding cl that interacts with the core polymerase to ensure its processivity, and (iii) the seven-subunit cl loader complex that loads β2 onto primer-template junctions and interacts with the α polymerase subunit of the core and the DnaB helicase to organize the two (or three) core polymerases. Here, we review the structures of the enzymatic components of replisomes, and the protein-protein and protein-DNA interactions that ensure they remain intact while undergoing substantial dynamic changes as they function to copy both the leading and lagging strands simultaneously during coordinated replication.

Genetic Encoding of para-Pentafluorosulfanyl Phenylalanine: A Highly Hydrophobic and Strongly Electronegative Group for Stable Protein Interactions

Publisher: American Chemical Society (ACS)

Date: 25-09-2020

DOI: 10.1021/JACS.0C07976

Assignment of paramagnetic 15N-HSQC spectra by heteronuclear exchange spectroscopy

Publisher: Springer Science and Business Media LLC

Date: 10-11-2006

DOI: 10.1007/S10858-006-9098-6

Abstract: Paramagnetic metal ions in proteins provide a rich source of structural information, but the resonance assignments required to extract the information can be challenging. Here we demonstrate that paramagnetically shifted (15)N-HSQC cross-peaks can be assigned using N(Z)-exchange spectroscopy under conditions in which the paramagnetic form of the protein is in dynamic equilibrium with its diamagnetic form. Even slow exchange of specifically bound metal ions may be detected within the long lifetime of (15)N longitudinal magnetization of large proteins at high magnetic fields. Alternatively, the exchange can be accelerated using an excess of metal ions. In the resulting exchange spectra, paramagnetic (15)N resonances become visible for residues that are not directly observed in a conventional (15)N-HSQC spectrum due to paramagnetic (1)H(N) broadening. The experiments are illustrated by the 30 kDa lanthanide-binding epsilon186/theta complex of DNA polymerase III in the presence of sub-stoichiometric amounts of Dy(3+) or a mixture of Dy(3+) and La(3+).

Disproportionation of a model chromium(V) complex causes extensive chromium(III)-DNA binding in vitro

Publisher: American Chemical Society (ACS)

Date: 13-07-2001

DOI: 10.1021/TX010077G

Abstract: The first direct evidence for the role of Cr(V) complexes in the formation of potentially mutagenic Cr(III)-DNA adducts has been obtained. A model complex for the stabilized Cr(V) species formed in Cr(VI)-treated cells, [Cr(V)O(ehba)(2)]-[ehba = 2-ethyl-2-hydroxybutanoato(2-)], rapidly disproportionates in HEPES buffers at pH 7.4 [3 Cr(V) --> 2 Cr(VI) + Cr(III)], and the formed Cr(III) species undergo efficient ionic binding to DNA, followed by slower covalent binding. The extent of Cr(III)-DNA binding significantly exceeds that caused by [Cr(III)(OH(2))(6)](3+) or by the Cr(III) products of Cr(VI) reductions under similar conditions. The Cr(III)-DNA binding can be dramatically reduced by the ability of the reaction medium (e.g., phosphate buffer) to form complexes with Cr(III) during and after the disproportionation reaction. A mechanism of Cr(III)-DNA binding caused by Cr(V) disproportionation has been proposed on the basis of stoichiometric and kinetic studies.

E. coli DNA replication in the absence of free β clamps

Publisher: Wiley

Date: 25-03-2011

DOI: 10.1038/EMBOJ.2011.84

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli

Publisher: Proceedings of the National Academy of Sciences

Date: 31-03-1998

DOI: 10.1073/PNAS.95.7.3472

Abstract: The structure of the proline-specific aminopeptidase (EC 3.4.11.9 ) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-Å resolution, for a dipeptide-inhibited complex at 2.3-Å resolution, and for a low-pH inactive form at 2.7-Å resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.

Backbone Assignment of Fully Protonated Solid Proteins by¹H Detection and Ultrafast Magic-Angle-Spinning NMR Spectroscopy

Publisher: Wiley

Date: 28-09-2012

DOI: 10.1002/ANIE.201203124

Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex

Publisher: Oxford University Press (OUP)

Date: 24-05-2015

DOI: 10.1093/NAR/GKV527

Structure Determination of Protein−Ligand Complexes by Transferred Paramagnetic Shifts

Publisher: American Chemical Society (ACS)

Date: 13-09-2006

DOI: 10.1021/JA063584Z

Abstract: Rational drug design depends on the knowledge of the three-dimensional (3D) structure of complexes between proteins and lead compounds of low molecular weight. A novel nuclear magnetic resonance (NMR) spectroscopy strategy based on the paramagnetic effects from lanthanide ions allows the rapid determination of the 3D structure of a small ligand molecule bound to its protein target in solution and, simultaneously, its location and orientation with respect to the protein. The method relies on the presence of a lanthanide ion in the protein target and on fast exchange between bound and free ligand. The binding affinity of the ligand and the paramagnetic effects experienced in the bound state are derived from concentration-dependent (1)H and (13)C spectra of the ligand at natural isotopic abundance. Combined with prior knowledge of the crystal or solution structure of the protein and of the magnetic susceptibility tensor of the lanthanide ion, the paramagnetic data define the location and orientation of the bound ligand molecule with respect to the protein from simple 1D NMR spectra. The method was verified with the ternary 30 kDa complex between the lanthanide-labeled N-terminal domain of the epsilon exonuclease subunit from the Escherichia coli DNA polymerase III, the subunit theta, and thymidine. The binding mode of thymidine was found to be very similar to that of thymidine monophosphate present in the crystal structure.

Bacterial Sliding Clamp Inhibitors that Mimic the Sequential Binding Mechanism of Endogenous Linear Motifs

Publisher: American Chemical Society (ACS)

Date: 28-05-2015

DOI: 10.1021/ACS.JMEDCHEM.5B00232

Abstract: The bacterial DNA replication machinery presents new targets for the development of antibiotics acting via novel mechanisms. One such target is the protein-protein interaction between the DNA sliding cl and the conserved peptide linear motifs in DNA polymerases. We previously established that binding of linear motifs to the Escherichia coli sliding cl occurs via a sequential mechanism that involves two subsites (I and II). Here, we report the development of small-molecule inhibitors that mimic this mechanism. The compounds contain tetrahydrocarbazole moieties as "anchors" to occupy subsite I. Functional groups appended at the tetrahydrocarbazole nitrogen bind to a channel gated by the side chain of M362 and lie at the edge of subsite II. One derivative induced the formation of a new binding pocket, termed subsite III, by rearrangement of a loop adjacent to subsite I. Discovery of the extended binding area will guide further inhibitor development.

Fast Structure-Based Assignment of ¹⁵N HSQC Spectra of Selectively ¹⁵N-Labeled Paramagnetic Proteins

Publisher: American Chemical Society (ACS)

Date: 13-02-2004

DOI: 10.1021/JA039339M

Abstract: A novel strategy for fast NMR resonance assignment of (15)N HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively (15)N-labeled s les. Comparison of sensitive undecoupled (15)N HSQC spectra recorded of paramagnetic and diamagnetic s les yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole-dipole relaxation, and residual dipolar coupling. Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center. The method is demonstrated with the 30 kDa complex between the N-terminal domain of the epsilon subunit and the theta subunit of Escherichia coli DNA polymerase III. The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy.

An isogeometric boundary element method for elastostatic analysis: 2D implementation aspects

Publisher: Elsevier BV

Date: 03-2013

DOI: 10.1016/J.COMPSTRUC.2012.12.021

Conservation of Eubacterial Replicases

Publisher: Wiley

Date: 06-2005

DOI: 10.1080/15216540500138246

Abstract: The last 15 years of effort in understanding bacterial DNA replication and repair has identified that the donut shaped beta2 sliding cl is harnessed by very functionally different DNA polymerases throughout the lifecycle of the bacterial cell. Remarkably, the sites of binding of these polymerases, in most cases, appear to be the same shallow pocket on the beta dimer. In every case, binding of beta2 by the polymerase enhances their processivity of DNA synthesis. This binding site is also the same point of interaction between beta2 and the cl loader complex, which binds beta2, opens and places it onto the DNA strand and then vacates the site. Beta2 may also be involved in the initiation of DNA replication again via contact through this same site. While much of the research effort has focused on Escherichia coli and Bacillus subtilis, conservation of this complex system is becoming apparent in erse bacteria.

Efficient χ-tensor determination and NH assignment of paramagnetic proteins

Publisher: Springer Science and Business Media LLC

Date: 06-2006

DOI: 10.1007/S10858-006-9002-4

Abstract: Anisotropic magnetic susceptibility tensors chi of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the chi-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D 15N-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable chi-tensor anisotropy parameters from 2D spectra of uniformly 15N-labeled proteins of fairly high molecular weight. Ex les are shown for the 185-residue N-terminal domain of the subunit epsilon from E. coli DNA polymerase III in complex with the subunit theta and La3+ in its diamagnetic and Dy3+, Tb3+, and Er3+ in its paramagnetic form.

Strain smoothing in FEM and XFEM

Publisher: Elsevier BV

Date: 12-2010

DOI: 10.1016/J.COMPSTRUC.2008.07.006

Stabilization of Native Protein Fold by Intein-Mediated Covalent Cyclization

Publisher: Elsevier BV

Date: 03-2005

DOI: 10.1016/J.JMB.2004.12.037

Abstract: A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.

Integron-associated Mobile Gene Cassettes Code for Folded Proteins: The Structure of Bal32a, a New Member of the Adaptable α+β Barrel Family

Publisher: Elsevier BV

Date: 03-2005

DOI: 10.1016/J.JMB.2004.12.035

Abstract: The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to s le the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a s le of eight cassette-encoded genes with no known homologs five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct s ling of mobile DNA as a route for the discovery of novel proteins.

AtfA, a new factor in global regulation of transcription in Acinetobacter spp

Publisher: Wiley

Date: 31-07-2014

DOI: 10.1111/MMI.12723

Abstract: Acinetobacter species are widely distributed bacteria in the environment, and have recently gained notoriety as opportunistic nosocomial pathogens. Here we characterize a novel RNA polymerase-interacting protein named acidic transcription factor A, AtfA. It is small and highly acidic, and is widely distributed throughout the γ proteobacteria, including other significant pathogens in the genera Moraxella, Pseudomonas, Legionella and Vibrio. In the model species A. baylyi ADP1, deletion of atfA significantly affects expression of over 500 genes, resulting in a large cell phenotype, reduced cell fitness, impaired biofilm formation and twitching motility, and increased sensitivity to antibiotics. Deletion of atfA also causes dramatically enhanced sensitivity to ethanol, which is an important growth promoter and virulence factor in Acinetobacter spp. The results suggest that auxiliary factors of RNA polymerase with important biological roles remain to be discovered.

Measurement of dissociation constants of high-molecular weight protein–protein complexes by transferred 15N-relaxation

Publisher: Springer Science and Business Media LLC

Date: 28-03-2007

DOI: 10.1007/S10858-007-9147-9

Abstract: The use of (15)N-relaxation data for determination of the dissociation constant of a protein-protein complex is proposed for the situation where a (15)N-labeled protein is bound to an unlabeled protein of high molecular weight, and the chemical exchange between bound and free protein is fast on the NMR time scale. The approach is shown to be suitable for estimating dissociation constants in the micromolar to millimolar range, using protein solutions at relatively low concentration. An ex le is shown for the interaction between two subunits from the Escherichia coli DNA polymerase III complex, involving a (15)N-labeled fragment of the C-terminal domain of the tau subunit (15 kDa) and the unlabeled alpha subunit (130 kDa).

Site-Specific Labelling of Proteins with a Rigid Lanthanide-Binding Tag

Publisher: Wiley

Date: 06-07-2006

DOI: 10.1002/CBIC.200600142

Abstract: This paper describes a generic method for the site-specific attachment of lanthanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Escherichia coli arginine repressor. Complexes with Y(3+), Tb(3+), Dy(3+), Ho(3+), Er(3+), Tm(3+) and Yb(3+) ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisotropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

Two- and three-dimensional isogeometric cohesive elements for composite delamination analysis

Publisher: Elsevier BV

Date: 04-2014

DOI: 10.1016/J.COMPOSITESB.2013.12.018

Synthesis and Applications of Covalent Protein-DNA Conjugates

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/CH09164

Abstract: Synthetic protein-DNA conjugates are valuable tools with applications in fields including nanobiotechnology, bioanalytical chemistry, and molecular diagnostics, and various synthetic methods for their production have been developed during the past three decades. The present article reviews current methodologies for the synthesis of covalent protein-DNA conjugates with particular focus on the regiospecificity and stoichiometry of these reactions.

Synthesis and properties of crosslinked recombinant pro-resilin

Publisher: Springer Science and Business Media LLC

Date: 10-2005

DOI: 10.1038/NATURE04085

Abstract: Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage it is best known for its roles in insect flight and the remarkable jumping ability of fleas and spittle bugs. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast s les of this biomaterial may enable its use in situ for both industrial and biomedical applications.

Structural characterisation of the complete cycle of sliding clamp loading inEscherichia coli

Publisher: Cold Spring Harbor Laboratory

Date: 21-07-2023

DOI: 10.1101/2023.07.20.549978

Abstract: Ring-shaped DNA sliding cl s are essential for DNA replication and genome maintenance. Cl s need to be opened or trapped open and chaperoned onto DNA by cl loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place cl s around DNA remains limited. Here, we present a series of six structures of the Escherichia coli CLC bound to an open or closed cl on and off a primer-template DNA that represent all intermediates in the cl loading process. We show that the ATP-bound CLC first binds to a cl , then constricts to hold onto it. The CLC then expands to open the cl with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing ATP hydrolysis and cl closing around DNA. Although both yeast and E. coli CLCs open cl s by crab claw-like motions, they do it by the CLC expanding in opposite directions. These structures provide critical high-resolution snapshots of cl loading by the E. coli CLC, revealing how the molecular machine works.

Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

Publisher: Springer Science and Business Media LLC

Date: 20-02-2015

DOI: 10.1038/NCOMMS7253

Abstract: Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator ( Icos ). When Roquin is absent or mutated (Roquin san ), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3′ end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the ‘ san’ mutation and Roquin’s ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.

Replication termination in Escherichia coli: Structure and antihelicase activity of the Tus-Ter complex

Publisher: American Society for Microbiology

Date: 09-2005

DOI: 10.1128/MMBR.69.3.501-526.2005

Abstract: The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein- Ter DNA complex. A replication fork can pass through a Tus- Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus- Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus- Ter and the helicase that prevents further DNA unwinding, while another is that the Tus- Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus- Ter complex with information available from the Tus- TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus- Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process.

Author response: Single-molecule visualization of fast polymerase turnover in the bacterial replisome

Publisher: eLife Sciences Publications, Ltd

Date: 15-04-2017

DOI: 10.7554/ELIFE.23932.027

Linear elastic fracture simulation directly from CAD: 2D NURBS-based implementation and role of tip enrichment

Publisher: Springer Science and Business Media LLC

Date: 30-09-2016

DOI: 10.1007/S10704-016-0153-3

The ROQUIN family of proteins localizes to stress granules via the ROQ domain and binds target mRNAs

Publisher: Wiley

Date: 19-04-2010

DOI: 10.1111/J.1742-4658.2010.07628.X

Abstract: Roquin is an E3 ubiquitin ligase with a poorly understood but essential role in preventing T-cell-mediated autoimmune disease and in microRNA-mediated repression of inducible costimulator (Icos) mRNA. Roquin and its mammalian paralogue membrane-associated nucleic acid binding protein (MNAB) define a protein family distinguished by an approximately 200 amino acid domain of unknown function, ROQ, that is highly conserved from mammals to invertebrates and is flanked by a RING-1 zinc finger and a CCCH zinc finger. Here we show that human, Drosophila and Caenorhabditis elegans Roquin and human MNAB localize to the cytoplasm and upon stress are concentrated in stress granules, where stalled mRNA translation complexes are stored. The ROQ domain is necessary and sufficient for localization to arsenite-induced stress granules and to induce these structures upon overexpression, and is required to trigger Icos mRNA decay. Gel-shift, SPR and footprinting studies show that an N-terminal fragment centred on the ROQ domain binds RNA from the Icos 3'-untranslated region comprising the minimal sequence for Roquin-mediated repression, adjacent to the miR-101 sequence complementarity. These findings identify Roquin as an RNA-binding protein and establish a specific function for the ROQ protein domain in mRNA homeostasis. Structured digital abstract * MINT-7711163: TIA-1 (uniprotkb:P31483) and Roquin (uniprotkb:Q4VGL6) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711475: RLE-1 (uniprotkb:O45962) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711487: DmRoquin (uniprotkb:Q9VV48) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711447, MINT-7711460: MNAB (uniprotkb:Q9HBD1) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711176: eIF3 (uniprotkb:P55884) and Roquin (uniprotkb:Q4VGL6) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711192: DCP1A (uniprotkb:Q9NPI6) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416).

Architecture tradeoffs of integrating a mesh generator to partition of unity enriched object-oriented finite element software

Publisher: River Publishers

Date: 2007

DOI: 10.3166/REMN.16.237-258

Modelling interfacial cracking with non-matching cohesive interface elements

Publisher: Springer Science and Business Media LLC

Date: 12-07-2016

DOI: 10.1007/S00466-016-1314-Y

Sequence-Specific and Stereospecific Assignment of Methyl Groups Using Paramagnetic Lanthanides

Publisher: American Chemical Society (ACS)

Date: 12-10-2007

DOI: 10.1021/JA0744753

Abstract: Pseudocontact shifts (PCSs) induced by a site-specifically bound paramagnetic lanthanide ion are shown to provide fast access to sequence-specific resonance assignments of methyl groups in proteins of known three-dimensional structure. Stereospecific assignments of Val and Leu methyls are obtained as well as resonance assignments of all other methyls, including Met epsilonCH3 groups. No prior assignments of the diamagnetic protein are required nor are experiments that transfer magnetization between the methyl groups and the protein backbone. Methyl Cz-exchange experiments were designed to provide convenient access to PCS measurements in situations where a paramagnetic lanthanide is in exchange with a diamagnetic lanthanide. In the absence of exchange, simultaneous 13C-HSQC assignments and PCS measurements are delivered by the newly developed program Possum. The approaches are demonstrated with the complex between the N-terminal domain of the subunit epsilon and the subunit theta of the Escherichia coli DNA polymerase III.

Kinetic and crystallographic analysis of mutant Escherichia coli aminopeptidase P: Insights into substrate recognition and the mechanism of catalysis

Publisher: American Chemical Society (ACS)

Date: 27-12-2006

DOI: 10.1021/BI0518904

Abstract: Aminopeptidase P (APPro) is a manganese-dependent enzyme that cleaves the N-terminal amino acid from polypeptides where the second residue is proline. APPro shares a similar fold, substrate specificity, and catalytic mechanism with methionine aminopeptidase and prolidase. To investigate the roles of conserved residues at the active site, seven mutant forms of APPro were characterized kinetically and structurally. Mutation of in idual metal ligands selectively abolished binding of either or both Mn(II) atoms at the active site, and none of these metal-ligand mutants had detectable catalytic activity. Mutation of the conserved active site residues His243 and His361 revealed that both are required for catalysis. We propose that His243 stabilizes substrate binding through an interaction with the carbonyl oxygen of the requisite proline residue of a substrate and that His361 stabilizes substrate binding and the gem-diol catalytic intermediate. Sequence, structural, and kinetic analyses reveal that His350, conserved in APPro and prolidase but not in methionine aminopeptidase, forms part of a hydrophobic binding pocket that gives APPro its proline specificity. Further, peptides in which the required proline residue is replaced by N-methylalanine or alanine are cleaved by APPro, but they are extremely poor substrates due to a loss of interactions between the prolidyl ring of the substrate and the hydrophobic proline-binding pocket.

A two-dimensional Isogeometric Boundary Element Method for elastostatic analysis

Publisher: Elsevier BV

Date: 02-2012

DOI: 10.1016/J.CMA.2011.08.008

Characterization of Gibberellin Receptor Mutants of Barley (Hordeum vulgare L.)

Publisher: Elsevier BV

Date: 03-2008

DOI: 10.1093/MP/SSN002

Abstract: The sequence of Gid1 (a gene for a gibberellin (GA) receptor from rice) was used to identify a putative orthologue from barley. This was expressed in E. coli, and produced a protein that was able to bind GA in vitro with both structural specificity and saturability. Its potential role in GA responses was investigated using barley mutants with reduced GA sensitivity (gse1 mutants). Sixteen different gse1 mutants each carried a unique nucleotide substitution in this sequence. In all but one case, these changes resulted in single amino acid substitutions, and, for the remaining mutant, a substitution in the 5' untranslated region of the mRNA is proposed to interfere with translation initiation. There was perfect linkage in segregating populations between new mutant alleles and the gse1 phenotype, leading to the conclusion that the putative GID1 GA receptor sequence in barley corresponds to the Gse1 locus. Determination of endogenous GA contents in one of the mutants revealed enhanced accumulation of bioactive GA(1), and a deficit of C(20) GA precursors. All of the gse1 mutants had reduced sensitivity to exogenous GA(3), and to AC94377 (a GA analogue) at concentrations that are normally 'saturating', but, at much higher concentrations, there was often a considerable response. The comparison between barley and rice mutants reveals interesting differences between these two cereal species in GA hormonal physiology.

Protein – Protein Interactions in the Eubacterial Replisome

Publisher: Wiley

Date: 2005

DOI: 10.1080/15216540500058956

Abstract: Replication of genomic DNA is a universal process that proceeds in distinct stages, from initiation to elongation and finally to termination. Each stage involves multiple stable or transient interactions between protein subunits with functions that are more or less conserved in all organisms. In Escherichia coli, initiation of bidirectional replication at the origin (oriC) occurs through the concerted actions of the DnaA replication initiator protein, the hexameric DnaB helicase, the DnaC?helicase loading partner and the DnaG primase, leading to establishment of two replication forks. Elongation of RNA primers at each fork proceeds simultaneously on both strands by actions of the multimeric replicase, DNA polymerase III holoenzyme. The fork that arrives first in the terminus region is halted by its encounter with a correctly-oriented complex of the Tus replication terminator protein bound at one of several Ter sites, where it is trapped until the other fork arrives. We summarize current understanding of interactions among the various proteins that act in the different stages of replication of the chromosome of E. coli, and make some comparisons with the analogous proteins in Bacillus subtilis and the coliphages T4 and T7.

Effect of protein stabilization on charge state distribution in positive- and negative-ion electrospray ionization mass spectra

Publisher: American Chemical Society (ACS)

Date: 09-2007

DOI: 10.1016/J.JASMS.2007.06.004

Abstract: Changes in protein conformation are thought to alter charge state distributions observed in electrospray ionization mass spectra (ESI-MS) of proteins. In most cases, this has been demonstrated by unfolding proteins through acidification of the solution. This methodology changes the properties of the solvent so that changes in the ESI-MS charge envelopes from conformational changes are difficult to separate from the effects of changing solvent on the ionization process. A novel strategy is presented enabling comparison of ESI mass spectra of a folded and partially unfolded protein of the same amino acid sequence subjected to the same experimental protocols and conditions. The N-terminal domain of the Escherichia coli DnaB protein was cyclized by in vivo formation of an amide bond between its N- and C-termini. The properties of this stabilized protein were compared with its linear counterpart. When the linear form was unfolded by decreasing pH, a charge envelope at lower m/z appeared consistent with the presence of a population of unfolded protein. This was observed in both positive-ion and negative-ion ESI mass spectra. Under the same conditions, this low m/z envelope was not present in the ESI mass spectrum of the stable cyclized form. The effects of changing the desolvation temperature in the ionization source of the Q-TOF mass spectrometer were also investigated. Increasing the desolvation temperature had little effect on positive-ion ESI mass spectra, but in negative-ion spectra, a charge envelope at lower m/z appeared, consistent with an increase in the abundance of unfolded protein molecules.

Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp

Publisher: American Society for Microbiology

Date: 06-2010

DOI: 10.1128/MMBR.00048-09

Abstract: Within the last 15 years, members of the bacterial genus Acinetobacter have risen from relative obscurity to be among the most important sources of hospital-acquired infections. The driving force for this has been the remarkable ability of these organisms to acquire antibiotic resistance determinants, with some strains now showing resistance to every antibiotic in clinical use. There is an urgent need for new antibacterial compounds to combat the threat imposed by Acinetobacter spp. and other intractable bacterial pathogens. The essential processes of chromosomal DNA replication, transcription, and cell ision are attractive targets for the rational design of antimicrobial drugs. The goal of this review is to examine the wealth of genome sequence and gene knockout data now available for Acinetobacter spp., highlighting those aspects of essential systems that are most suitable as drug targets. Acinetobacter spp. show several key differences from other pathogenic gammaproteobacteria, particularly in global stress response pathways. The involvement of these pathways in short- and long-term antibiotic survival suggests that Acinetobacter spp. cope with antibiotic-induced stress differently from other microorganisms.

Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit.

Publisher: American Society for Microbiology

Date: 15-06-2006

DOI: 10.1128/JB.01992-05

Abstract: The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the α, ε, and θ subunits. The θ subunit is the smallest and least understood subunit. The three-dimensional structure of θ in a complex with the unlabeled N-terminal domain of the ε subunit, ε186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy 3+ , Er 3+ , or Ho 3+ ) at the active site of ε186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of θ in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed θ show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free θ (Keniry et al., Protein Sci. 9: 721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of θ in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of ε186 on θ was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of θ are located about 15 Å or farther from the lanthanide ion in the active site of ε186, in agreement with the extensive biochemical data for the θ-ε system.

Front Cover: Rational Design of a 310‐Helical PIP‐Box Mimetic Targeting PCNA, the Human Sliding Clamp (Chem. Eur. J. 44/2018)

Publisher: Wiley

Date: 19-07-2018

DOI: 10.1002/CHEM.201803279

Disproportionation and nuclease activity of bis[2-ethyl-2-hydroxybutanoato(2-)]oxochromate(V) in neutral aqueous solutions

Publisher: American Chemical Society (ACS)

Date: 13-01-2000

DOI: 10.1021/IC990729C

Abstract: Complex 1, [Cr(V)O(ehba)2]- (ehba = 2-ethyl-2-hydroxybutanoate(2-)) is the most studied model compound of relevance to the biological activity of Cr(V) with regard to Cr-induced cancers. The first detailed kinetic study of disproportionation of 1 under neutral pH conditions (pH 6.0-8.0, [NaClO4] = 1.0 M, 37 degrees C) is reported. Kinetic data were collected by stopped-flow and conventional UV-vis spectroscopies and processed by the global analysis method. The disproportionation, which follows the stoichiometry 3Cr(V) --> 2Cr(VI) + Cr(III) (1), leads to release of 5 mol of H+/3 mol of Cr(V). Reaction 1 is accelerated by phosphate, but is not affected by acetate, HEPES, or Tris buffers. Initial rates of Cr(V) decay are directly proportional to [Cr(V)]0 (0.020-1.0 mM) they increase with an increase in the pH values and decrease in the presence of a large excess of ehba ligand. The first direct evidence for the formation of Cr(IV) intermediates in reaction 1 has been obtained however, their UV-vis spectral properties were different from those of the well-characterized Cr(IV)-ehba complexes. The Cr(III) products of reaction I in phosphate buffers differ from those in the other buffers. A mechanism is proposed for reaction 1 on the basis of kinetic modeling. Influences of the reaction time and conditions on the extent of plasmid DNA cleavage induced by 1 have been studied under conditions corresponding to those of the kinetic studies. A comparison of the kinetic and DNA cleavage results has shown that direct interaction of 1 with the phosphate backbone of DNA is the most likely first step in the mechanism of DNA cleavage in neutral media. Small additions of Mn(II) ((0.01-0.1)[Cr(V)]0) did not affect the rate and stoichiometry of reaction 1, but suppressed the formation of Cr(IV) intermediates (presumably due to the catalysis of Cr(IV) disproportionation). However, much higher concentrations of Mn(II) ((0.1-1.0)[Cr(V)]0) were required to inhibit DNA cleavage induced by 1. Thus, contrary to previous reports (Sugden, K. D. Wetterhahn, K. E. J. Am. Chem. Soc. 1996, 118, 10811-10818), inhibition by Mn(II) does not indicate a key role of Cr(IV) in Cr(V)-induced DNA cleavage.

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Publisher: Springer Science and Business Media LLC

Date: 27-01-2008

DOI: 10.1038/NSMB.1381

What is all this fuss about Tus? Comparison of recent findings from biophysical and biochemical experiments.

Publisher: Informa UK Limited

Date: 06-11-2017

DOI: 10.1080/10409238.2017.1394264

Abstract: Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a "replication fork trap" - found within a chromosomal region where forks are allowed to enter but not leave - is set by the protein-DNA roadblock Tus-Ter. The exact sequence of events by which Tus-Ter blocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein-protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus-Ter interactions at the nonpermissive face - interactions that result in a highly stable "locked" complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus-Ter mechanism - leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA-protein system further.

Discovery of Lead Compounds Targeting the Bacterial Sliding Clamp Using a Fragment-Based Approach

Publisher: American Chemical Society (ACS)

Date: 18-03-2014

DOI: 10.1021/JM500122R

Abstract: The bacterial sliding cl (SC), also known as the DNA polymerase III β subunit, is an emerging antibacterial target that plays a central role in DNA replication, serving as a protein-protein interaction hub with a common binding pocket to recognize linear motifs in the partner proteins. Here, fragment-based screening using X-ray crystallography produced four hits bound in the linear-motif-binding pocket of the Escherichia coli SC. Compounds structurally related to the hits were identified that inhibited the E. coli SC and SC-mediated DNA replication in vitro. A tetrahydrocarbazole derivative emerged as a promising lead whose methyl and ethyl ester prodrug forms showed minimum inhibitory concentrations in the range of 21-43 μg/mL against representative Gram-negative and Gram-positive bacteria species. The work demonstrates the utility of a fragment-based approach for identifying bacterial sliding cl inhibitors as lead compounds with broad-spectrum antibacterial activity.

Solution structure of Domains IVa and V of the τ subunit of Escherichia coli DNA polymerase III and interaction with the α subunit

Publisher: Oxford University Press (OUP)

Date: 22-04-2007

DOI: 10.1093/NAR/GKM080

Strand separation establishes a sustained lock at the Tus–Ter replication fork barrier

Publisher: Springer Science and Business Media LLC

Date: 06-07-2015

DOI: 10.1038/NCHEMBIO.1857

Abstract: The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.

Nanometer-Scale Distance Measurements in Proteins Using Gd³⁺ Spin Labeling

Publisher: American Chemical Society (ACS)

Date: 10-06-2010

DOI: 10.1021/JA1015662

Abstract: Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd(3+) spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and tau(C)14, labeled at strategically selected sites with either two nitroxides or two Gd(3+) spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd(3+) tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (approximately 9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd(3+)-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd(3+)-Gd(3+) distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein tau(C)14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd(3+)-Gd(3+) distance distribution with a maximum at 3.4 nm. The Gd(3+)-Gd(3+) distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd(3+) labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection.

Monomeric solution structure of the helicase‐binding domain of Escherichia coli DnaG primase

Publisher: Wiley

Date: 28-09-2006

DOI: 10.1111/J.1742-4658.2006.05495.X

Abstract: DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1-171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1-171 of the E. coli DnaB helicase with significant affinity.

The unstructed C-terminus of the τ subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the α subunit

Publisher: Oxford University Press (OUP)

Date: 13-03-2007

DOI: 10.1093/NAR/GKM079

Preliminary X-ray crystallographic and NMR studies on the exonuclease domain of the ε subunit of Escherichia coli DNA polymerase III

Publisher: Elsevier BV

Date: 08-2000

DOI: 10.1006/JSBI.2000.4291

Structural basis for proofreading during replication of the Escherichia coli chromosome.

Publisher: Elsevier BV

Date: 04-2002

DOI: 10.1016/S0969-2126(02)00738-4

Abstract: The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease. Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate. The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily. The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site. These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA.

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode

Publisher: Wiley

Date: 22-02-2013

DOI: 10.1038/EMBOJ.2012.347

Bound or Free: Interaction of the C-Terminal Domain of Escherichia coli Single-Stranded DNA-Binding Protein (SSB) with the Tetrameric Core of SSB

Publisher: American Chemical Society (ACS)

Date: 18-03-2014

DOI: 10.1021/BI5001867

Abstract: Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

Recycling of single-stranded DNA-binding protein by the bacterial replisome

Publisher: Oxford University Press (OUP)

Date: 15-02-2019

DOI: 10.1093/NAR/GKZ090

Defining the structural basis of human plasminogen binding by streptococcal surface enolase

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1074/JBC.M109.004317

Amino-acid Type Identification in 15N-HSQC Spectra by Combinatorial Selective 15N-labelling

Publisher: Springer Science and Business Media LLC

Date: 2006

DOI: 10.1007/S10858-005-5021-9

Abstract: The efficiency of cell-free protein synthesis combined with combinatorial selective 15N-labelling provides a method for the rapid assignment of 15N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five 15N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the tau subunit of the Escherichia coli DNA polymerase III holoenzyme, tauC16 and tauC14. Since each of the five 15N-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled s les, spectral overlap was much reduced. All 15N-HSQC cross-peaks of the backbone amides could be assigned to the correct amino-acid type. Availability of the residue-type information greatly assisted the evaluation of the changes in chemical shifts observed for corresponding residues in tauC16 vs. those in tauC14, and the analysis of the structure and mobility of the C-terminal residues present in tauC16 but not in tauC14.

Single-molecule studies of fork dynamics in Escherichia coli DNA replication (Nature Structural and Molecular Biology (2008) 15, (170-176))

Publisher: Springer Science and Business Media LLC

Date: 08-2107

DOI: 10.1038/NSMB0908-998A

An implicit potential method along with a meshless technique for incompressible fluid flows for regular and irregular geometries in 2D and 3D

Publisher: Elsevier BV

Date: 04-2017

DOI: 10.1016/J.ENGANABOUND.2017.01.009

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

Publisher: Cold Spring Harbor Laboratory

Date: 19-08-2021

DOI: 10.1101/2021.08.18.456904

Abstract: Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long-lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP-dependent motor protein distantly related to helicases. It was first identified in the model Gram-positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work we have undertaken the first comprehensive investigation of the phylogenetic ersity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. Therefore, HelD proteins represent an important new class of transcription factor associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

Nuclease dead Cas9 is a programmable roadblock for DNA replication

Publisher: Springer Science and Business Media LLC

Date: 16-09-2019

DOI: 10.1038/S41598-019-49837-Z

Abstract: Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro . We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro .

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

Publisher: Wiley

Date: 12-2021

DOI: 10.1002/MBO3.1251

Abstract: Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long‐lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP‐dependent motor proteins distantly related to helicases. It was first identified in the model Gram‐positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram‐positive bacteria. In this work, we have undertaken the first comprehensive investigation of the phylogenetic ersity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram‐negative bacteria. In summary, HelD proteins represent an important new class of transcription factors associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

Lanthanide Labeling Offers Fast NMR Approach to 3D Structure Determinations of Protein−Protein Complexes

Publisher: American Chemical Society (ACS)

Date: 25-02-2006

DOI: 10.1021/JA057008Z

Abstract: A novel nuclear magnetic resonance (NMR) strategy based on labeling with lanthanides achieves rapid determinations of accurate three-dimensional (3D) structures of protein-protein complexes. The method employs pseudocontact shifts (PCS) induced by a site-specifically bound lanthanide ion to anchor the coordinate system of the magnetic susceptibility tensor in the molecular frames of the two molecules. Simple superposition of the tensors detected in the two protein molecules brings them together in a 3D model of the protein-protein complex. The method is demonstrated with the 30 kDa complex between two subunits of Escherichia coli polymerase III, comprising the N-terminal domain of the exonuclease subunit epsilon and the subunit theta. The 3D structures of the in idual molecules were docked based on a limited number of PCS observed in 2D 15N-heteronuclear single quantum coherence spectra. Degeneracies in the mutual orientation of the protein structures were resolved by the use of two different lanthanide ions, Dy3+ and Er3+.

Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: A model derived from DNA-binding studies of mutant proteins by surface plasmon resonance

Publisher: American Chemical Society (ACS)

Date: 12-09-2000

DOI: 10.1021/BI001174W

Abstract: The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K(D)) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The K(D) measured for the binding of wild type and His(6)-Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased K(D) by 200-300-fold, whereas the A173T substitution increased K(D) by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein.

NMR Detection of Protein ¹⁵N Spins near Paramagnetic Lanthanide Ions

Publisher: American Chemical Society (ACS)

Date: 29-12-2006

DOI: 10.1021/JA066995O

Hybrid Methods for Protein Structure Prediction

Publisher: Wiley

Date: 07-09-2010

DOI: 10.1002/9780470882207.CH12

Programming the material point method in Julia

Publisher: Elsevier BV

Date: 03-2017

DOI: 10.1016/J.ADVENGSOFT.2017.01.008

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

Publisher: American Chemical Society (ACS)

Date: 16-12-2011

DOI: 10.1021/JA106416G

Abstract: The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

Isogeometric analysis suitable trivariate NURBS representation of composite panels with a new offset algorithm

Publisher: Elsevier BV

Date: 10-2014

DOI: 10.1016/J.CAD.2014.05.004

The E. coli helicase does not use ATP during replication

Publisher: Cold Spring Harbor Laboratory

Date: 07-07-2021

DOI: 10.1101/2021.07.07.451541

Abstract: The replisome is responsible for replication of DNA in all domains of life, with several of its in idual enzyme components relying on hydrolysis of nucleoside triphosphates to provide energy for replisome function. Half a century of biochemical studies have demonstrated a dependence on ATP as an energy source for helicases to unwind duplex DNA during replication. Through single-molecule visualization of DNA replication by the Escherichia coli replisome, we demonstrate that the DnaB helicase does not rely on hydrolysis of ATP (or any ribo-NTPs) in the context of the elongating replisome. We establish that nucleotide incorporation by the leading-strand polymerase is the main motor driving the replication process. Polymerases provide the energy for helicase-mediated DNA unwinding during E. coli DNA replication.

EX1 hydrogen–deuterium exchange in an all-helical protein and its cyclized derivative at neutral pH

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1016/J.IJMS.2010.09.011

Development of a single-stranded DNA-binding protein fluorescent fusion toolbox

Publisher: Oxford University Press (OUP)

Date: 06-05-2020

DOI: 10.1093/NAR/GKAA320

Abstract: Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

Single-molecule visualization of fast polymerase turnover in the bacterial replisome

Publisher: eLife Sciences Publications, Ltd

Date: 22-04-2017

DOI: 10.7554/ELIFE.23932

Abstract: The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding cl ), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled cl loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.

Isogeometric analysis: An overview and computer implementation aspects

Publisher: Elsevier BV

Date: 11-2015

DOI: 10.1016/J.MATCOM.2015.05.008

Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase

Publisher: Elsevier BV

Date: 02-2004

DOI: 10.1016/J.PEP.2003.10.001

Abstract: In Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E. coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage lambda-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5mM s le in 10mM phosphate, pH 6.05, 0.1M NaCl, recorded at 36 degrees C, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a=b=142.2A, c=192.1A, and diffracted beyond 2.7A resolution with synchrotron radiation.

Intramolecular binding mode of the C-terminus ofEscherichia colisingle-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

Publisher: Oxford University Press (OUP)

Date: 27-11-2013

DOI: 10.1093/NAR/GKT1238

Abstract: Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron density of the C-peptide. Here we show that SSB forms a monomer at pH 3.4, which is suitable for studies by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The OB-domain retains its 3D structure in the monomer, and the C-peptide is shown by nuclear Overhauser effects and lanthanide-induced pseudocontact shifts to bind to the OB-domain at a site that harbors ssDNA in the crystal structure of the SSB–ssDNA complex. 15N relaxation data demonstrate high flexibility of the polypeptide segment linking the C-peptide to the OB-domain and somewhat increased flexibility of the C-peptide compared with the OB-domain, suggesting that the C-peptide either retains high mobility in the bound state or is in a fast equilibrium with an unbound state.

An investigation into the interactions of gold nanoparticles and anti-arthritic drugs with macrophages, and their reactivity towards thioredoxin reductase

Publisher: Elsevier BV

Date: 2015

DOI: 10.1016/J.JINORGBIO.2014.09.013

Abstract: Gold(I) complexes are an important tool in the arsenal of established approaches for treating rheumatoid arthritis (RA), while some recent studies have suggested that gold nanoparticles (Au NPs) may also be therapeutically efficacious. These observations prompted the current biological studies involving gold(I) anti-RA agents and Au NPs, which are aimed towards improving our knowledge of how they work. The cytotoxicity of auranofin, aurothiomalate, aurothiosulfate and Au NPs towards RAW264.7 macrophages was evaluated using the MTT assay, with the former compound proving to be the most toxic. The extent of cellular uptake of the various gold agents was determined using graphite furnace atomic absorption spectrometry, while their distribution within macrophages was examined using microprobe synchrotron radiation X-ray fluorescence spectroscopy. The latter technique showed accumulation of gold in discrete regions of the cell, and co-localisation with sulfur in the case of cells treated with aurothiomalate or auranofin. Electrospray ionization mass spectrometry was used to characterize thioredoxin reductase (TrxR) in which the penultimate selenocysteine residue was replaced by cysteine. Mass spectra of solutions of TrxR and aurothiomalate, aurothiosulfate or auranofin showed complexes containing bare gold atoms bound to the protein, or protein adducts containing gold atoms retaining some of their initial ligands. These results support TrxR being an important target of gold(I) drugs used to treat RA, while the finding that Au NPs are incorporated into macrophages, but elicit little toxicity, indicates further exploration of their potential for treatment of RA is warranted.

DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies

Publisher: Oxford University Press (OUP)

Date: 17-06-2021

DOI: 10.1093/NAR/GKAB493

Abstract: In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the cl -loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.

Introduction to trifluoromethanesulfonates and trifluoromethanesulfonato-O Complexes

Publisher: Wiley

Date: 2007

DOI: 10.1002/9780470132555.CH68

Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin–resistant Staphylococcus aureus

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1016/J.BMCL.2018.09.038

Virtual and smoothed finite elements: A connection and its application to polygonal/polyhedral finite element methods

Publisher: Wiley

Date: 21-07-2015

DOI: 10.1002/NME.4965

Weakening the tight coupling between geometry and simulation in isogeometric analysis: From sub‐ and super‐geometric analysis to Geometry‐Independent Field approximaTion (GIFT)

Publisher: Wiley

Date: 30-03-2018

DOI: 10.1002/NME.5778

Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins

Publisher: American Chemical Society (ACS)

Date: 23-04-2004

DOI: 10.1021/BI036229J

Abstract: The sliding cl of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding cl , a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Crystal structures and biochemical characterization of DNA sliding clamps from three Gram-negative bacterial pathogens

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1016/J.JSB.2018.10.008

Abstract: Bacterial sliding cl s bind to DNA and act as protein-protein interaction hubs for several proteins involved in DNA replication and repair. The partner proteins all bind to a common pocket on sliding cl s via conserved linear peptide sequence motifs, which suggest the pocket as an attractive target for development of new antibiotics. Herein we report the X-ray crystal structures and biochemical characterization of β sliding cl s from the Gram-negative pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae. The structures reveal close similarity between the pathogen and Escherichia coli cl s and similar patterns of binding to linear cl -binding motif peptides. The results suggest that linear motif-sliding cl interactions are well conserved and an antibiotic targeting the sliding cl should have broad-spectrum activity against Gram-negative pathogens.

A Single Subunit Directs the Assembly of the Escherichia coli DNA Sliding Clamp Loader

Publisher: Elsevier BV

Date: 03-2010

DOI: 10.1016/J.STR.2010.01.009

A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli

Publisher: Elsevier BV

Date: 06-2006

DOI: 10.1016/J.CELL.2006.04.040

Abstract: During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 A from its normal position to bind in a cytosine-specific pocket on the surface of Tus.

Weak Alignment of Paramagnetic Proteins Warrants Correction for Residual CSA Effects in Measurements of Pseudocontact Shifts

Publisher: American Chemical Society (ACS)

Date: 12-11-2005

DOI: 10.1021/JA0564259

Abstract: Paramagnetic metal ions can induce molecular alignment with respect to the magnetic field. This alignment generates residual anisotropic chemical shifts (RACS) due to nonisotropic averaging over the molecular orientations. Using a 30 kDa protein-protein complex, the RACS effects are shown to be significant for heteronuclear spins with large chemical shift anisotropies, lanthanide ions with large anisotropic magnetic susceptibility tensors, and measurements at high magnetic field. Therefore, RACS must be taken into account when pseudocontact shifts are measured by comparison of chemical shifts observed between complexes with paramagnetic and diamagnetic lanthanide ions. The results are of particular importance when different pseudocontact shifts measured for the 1HN, 15N, and 13C' spins of a peptide group are used to restrain its orientation with respect to the electronic magnetic susceptibility tensor in structure calculations.

Fundamental solutions and dual boundary element methods for fracture in plane Cosserat elasticity

Publisher: The Royal Society

Date: 07-2015

DOI: 10.1098/RSPA.2015.0216

Abstract: In this paper, both singular and hypersingular fundamental solutions of plane Cosserat elasticity are derived and given in a ready-to-use form. The hypersingular fundamental solutions allow to formulate the analogue of Somigliana stress identity, which can be used to obtain the stress and couple-stress fields inside the domain from the boundary values of the displacements, microrotation and stress and couple-stress tractions. Using these newly derived fundamental solutions, the boundary integral equations of both types are formulated and solved by the boundary element method. Simultaneous use of both types of equations (approach known as the dual boundary element method (BEM)) allows problems where parts of the boundary are overlapping, such as crack problems, to be treated and to do this for general geometry and loading conditions. The high accuracy of the boundary element method for both types of equations is demonstrated for a number of benchmark problems, including a Griffith crack problem and a plate with an edge crack. The detailed comparison of the BEM results and the analytical solution for a Griffith crack and an edge crack is given, particularly in terms of stress and couple-stress intensity factors, as well as the crack opening displacements and microrotations on the crack faces and the angular distributions of stresses and couple-stresses around the crack tip.

Ultrasensitive detection of antibodies using a new Tus–Ter-lock immunoPCR system

Publisher: Royal Society of Chemistry (RSC)

Date: 2010

DOI: 10.1039/C002163F

Abstract: A system consisting of a protein LG coated surface for the capture of mammalian antibodies (target), and an antigen fused to Tus and stoichiometrically linked to a DNA template via the Tus-Ter-lock sequence allowed the ultrasensitive detection of 5.5 attomol of target by real-time immunoPCR in complex media.

Meshless methods: A review and computer implementation aspects

Publisher: Elsevier BV

Date: 12-2008

DOI: 10.1016/J.MATCOM.2008.01.003

Multiple oligomeric forms of Escherichia coli DnaB helicase revealed by electrospray ionisation mass spectrometry

Publisher: Wiley

Date: 11-12-2006

DOI: 10.1002/RCM.2818

Abstract: The Escherichia coli DnaB protein (DnaB(6)) is the hexameric helicase that unwinds genomic DNA so it can be copied by the DNA replication machinery. Loading of the helicase onto DNA requires interactions of DnaB(6) with six molecules of its loading partner protein, DnaC. Nano-electrospray ionisation mass spectrometry (nanoESI-MS) of mutant proteins was used to examine the roles of the residues Phe102 (F102) and Asp82 (D82) in the N-terminal domain of DnaB in the assembly of the hexamer. When the proteins were prepared in 1 M ammonium acetate containing magnesium and adenosine triphosphate (ATP) at pH 7.6, both hexameric and heptameric forms of wild-type and F102W, F102E and D82N mutant DnaBs were observed in mass spectra. The spectra of the D82N mutant also showed substantial amounts of a decameric species and small amounts of a dodecamer. In contrast, the F102H DnaB mutant was incapable of forming oligomers of order higher than the hexamer. Thus, although Phe102 is not the only determinant of hexamer assembly, this residue has a role in oligomerisation. NanoESI mass spectra were obtained of mixtures of DnaB(6) with DnaC. The DnaB(6)(DnaC)(6) complex (calculated M(r) 481 164) was observed only when the two proteins were present in equimolar amounts. The data are consistent with cooperative assembly of the complex. ESI mass spectra of mixtures containing DnaC and ATP showed that DnaC slowly hydrolysed ATP to ADP as indicated by ions corresponding to DnaC/ATP and DnaC/ADP complexes. These experiments show that E. coli DnaB can form a heptameric complex and that nanoESI-MS can be used to probe assembly of large (>0.5 MDa) macromolecular complexes.

Optimization of an Escherichia coli system for cell‐free synthesis of selectively ¹⁵N‐labelled proteins for rapid analysis by NMR spectroscopy

Publisher: Wiley

Date: 24-09-2004

DOI: 10.1111/J.1432-1033.2004.04346.X

Abstract: Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [15N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage lambdapR and pL promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen s les, each selectively labelled with a different 15N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15N-HSQC spectra of 13 of the s les. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [15N]Asp, where an enzyme in the cell extract efficiently converted Asp-->Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.

A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

Publisher: Oxford University Press (OUP)

Date: 02-03-2009

DOI: 10.1093/NAR/GKP092

Proteomic dissection of DNA polymerization

Publisher: Informa UK Limited

Date: 04-2006

DOI: 10.1586/14789450.3.2.197

Abstract: DNA polymerases replicate the genome by associating with a range of other proteins that enable rapid, high-fidelity copying of DNA. This complex of proteins and nucleic acids is termed the replisome. Proteins of the replisome must interact with other networks of proteins, such as those involved in DNA repair. Many of the proteins involved in DNA polymerization and the accessory proteins are known, but the array of proteins they interact with, and the spatial and temporal arrangement of these interactions, are current research topics. Mass spectrometry is a technique that can be used to identify the sites of these interactions and to determine the precise stoichiometries of binding partners in a functional complex. A complete understanding of the macromolecular interactions involved in DNA replication and repair may lead to discovery of new targets for antibiotics against bacteria and biomarkers for diagnosis of diseases, such as cancer, in humans.

Site-specific covalent attachment of DNA to proteins using a photoactivatable Tus–Ter complex

Publisher: Royal Society of Chemistry (RSC)

Date: 2009

DOI: 10.1039/B900905A

Abstract: Investigations into the photocrosslinking kinetics of the protein Tus with various bromodeoxyuridine-substituted Ter DNA variants highlight the potential use of this complex as a photoactivatable connector between proteins of interest and specific DNA sequences.

Chromium(VI) reduction by catechol(amine)s results in DNA cleavage in vitro: Relevance to chromium genotoxicity

Publisher: American Chemical Society (ACS)

Date: 26-04-2001

DOI: 10.1021/TX000229S

Abstract: Catechols are found extensively in nature both as essential biomolecules and as the byproducts of normal oxidative damage of amino acids and proteins. They are also present in cigarette smoke and other atmospheric pollutants. Here, the interactions of reactive species generated in Cr(VI)/catechol(amine) mixtures with plasmid DNA have been investigated to model a potential route to Cr(VI)-induced genotoxicity. Reduction of Cr(VI) by 3,4-dihydroxyphenylalanine (DOPA) (1), dopamine (2), or adrenaline (3) produces species that cause extensive DNA damage, but the products of similar reactions with catechol (4) or 4-tert-butylcatechol (5) do not damage DNA. The Cr(VI)/catechol(amine) reactions have been studied at low added H(2)O(2) concentrations, which lead to enhanced DNA cleavage with 1 and induce DNA cleavage with 4. The Cr(V) and organic intermediates generated by the reactions of Cr(VI) with 1 or 4 in the presence of H(2)O(2) were characterized by EPR spectroscopy. The detected signals were assigned to Cr(V)-catechol, Cr(V)-peroxo, and mixed Cr(V)-catechol-peroxo complexes. Oxygen consumption during the reactions of Cr(VI) with 1, 2, 4, and 5 was studied, and H(2)O(2) production was quantified. Reactions of Cr(VI) with 1 and 2, but not 4 and 5, consume considerable amounts of dissolved O(2), and give extensive H(2)O(2) production. Extents of oxygen consumption and H(2)O(2) production during the reaction of Cr(VI) with enzymatically generated 1 and N-acetyl-DOPA (from the reaction of Tyr and N-acetyl-Tyr with tyrosinase, respectively) were correlated with the DNA cleaving abilities of the products of these reactions. The reaction of Cr(VI) with enzymatically generated 1 produced significant amounts of H(2)O(2) and caused significant DNA damage, but the N-acetyl-DOPA did not. The extent of in vitro DNA damage is reduced considerably by treatment of the Cr(VI)/catechol(amine) mixtures with catalase, which shows that the DNA damage is H(2)O(2)-dependent and that the major reactive intermediates are likely to be Cr(V)-peroxo and mixed Cr(V)-catechol-peroxo complexes, rather than Cr(V)-catechol intermediates.

Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis

Publisher: Oxford University Press (OUP)

Date: 26-09-2006

DOI: 10.1093/NAR/GKL690

DNA Replication Is the Target for the Antibacterial Effects of Nonsteroidal Anti-Inflammatory Drugs

Publisher: Elsevier BV

Date: 04-2014

DOI: 10.1016/J.CHEMBIOL.2014.02.009

Abstract: Evidence suggests that some nonsteroidal anti-inflammatory drugs (NSAIDs) possess antibacterial properties with an unknown mechanism. We describe the in vitro antibacterial properties of the NSAIDs carprofen, bromfenac, and vedaprofen, and show that these NSAIDs inhibit the Escherichia coli DNA polymerase III β subunit, an essential interaction hub that acts as a mobile tether on DNA for many essential partner proteins in DNA replication and repair. Crystal structures show that the three NSAIDs bind to the sliding cl at a common binding site required for partner binding. Inhibition of interaction of the cl loader and/or the replicative polymerase α subunit with the sliding cl is demonstrated using an in vitro DNA replication assay. NSAIDs thus present promising lead scaffolds for novel antibacterial agents targeting the sliding cl .

Fragment-based discovery of inhibitors of the bacterial DnaG-SSB interaction

Publisher: MDPI AG

Date: 22-02-2018

DOI: 10.3390/ANTIBIOTICS7010014

Real-time single-molecule observation of rolling-circle DNA replication

Publisher: Oxford University Press (OUP)

Date: 20-01-2009

DOI: 10.1093/NAR/GKP006

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Publisher: American Chemical Society (ACS)

Date: 03-01-2013

DOI: 10.1007/S13361-012-0552-2

Abstract: Single-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)(70), one molecule of (dT)(35), or two molecules of (dT)(35)] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III.

Publisher: Wiley

Date: 29-12-2004

DOI: 10.1110/PS.04889604

Mhp182 (P102) binds fibronectin and contributes to the recruitment of plasmin(ogen) to the Mycoplasma hyopneumoniae cell surface

Publisher: Hindawi Limited

Date: 09-11-2012

DOI: 10.1111/J.1462-5822.2011.01702.X

Abstract: Mycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (K(D) ~ 76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with K(D) s of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence.

Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions

Publisher: Springer Science and Business Media LLC

Date: 07-2005

DOI: 10.1007/S10858-005-7946-4

Abstract: Cell-free protein synthesis systems provide facile access to proteins in a nascent state that enables formation of soluble, native protein-protein complexes even if one of the protein components is prone to self-aggregation and precipitation. Combined with selective isotope-labeling, this allows the rapid analysis of protein-protein interactions with few 15N-HSQC spectra. The concept is demonstrated with binary and ternary complexes between the chi, psi and gamma subunits of Escherichia coli DNA polymerase III: nascent, selectively 15N-labeled psi produced in the presence of chi resulted in a soluble, correctly folded chi-psi complex, whereas psi alone precipitated irrespective of whether gamma was present or not. The 15N-HSQC spectra showed that the N-terminal segment of psi is mobile in the chi-psi complex, yet important for its binding to gamma. The s le preparation was greatly enhanced by an autoinduction strategy, where the T7 RNA polymerase needed for transcription of a gene in a T7-promoter vector was produced in situ.

Isogeometric boundary element methods for three dimensional static fracture and fatigue crack growth

Publisher: Elsevier BV

Date: 04-2017

DOI: 10.1016/J.CMA.2016.05.038

Incorporation of chlorinated analogues of aliphatic amino acids during cell-free protein synthesis

Publisher: Royal Society of Chemistry (RSC)

Date: 2011

DOI: 10.1039/C0CC02879G

Abstract: 3-Chloro-Abu and 4-chloro-Nva are biosynthetically incorporated into E. coli peptidyl-Pro cis-trans isomerase B, as substitutes for Val and Leu, respectively. The extent of incorporation is up to ~90%, and substituted protein is catalytically active. By contrast, 4-chloro-Val is not an effective replacement for Ile.

Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain

Publisher: Oxford University Press (OUP)

Date: 10-04-2013

DOI: 10.1093/NAR/GKT162

Rational Design of a 310 -Helical PIP-Box Mimetic Targeting PCNA, the Human Sliding Clamp

Publisher: Wiley

Date: 23-07-2018

DOI: 10.1002/CHEM.201803280

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Publisher: Wiley

Date: 2002

DOI: 10.1110/PS.PS.27702

In Vivo Protein Cyclization Promoted by a Circularly Permuted Synechocystis sp. PCC6803 DnaB Mini-intein

Publisher: Elsevier BV

Date: 03-2002

DOI: 10.1074/JBC.M110303200

Escherichia coli PII protein: purification, crystallization and oligomeric structure

Publisher: Wiley

Date: 17-01-1994

DOI: 10.1016/0014-5793(94)80203-3

Abstract: The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction. The crystals belong to space group P6(3) with a = b = 61.6 A, c = 56.3 A and Vm of 2.5 for one subunit in the asymmetric unit. A low-resolution electron density map showed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 +/- 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.

Probing molecular choreography through single-molecule biochemistry

Publisher: Springer Science and Business Media LLC

Date: 12-2015

DOI: 10.1038/NSMB.3119

Abstract: Single-molecule approaches are having a dramatic impact on views of how proteins work. The ability to observe molecular properties at the single-molecule level allows characterization of subpopulations and acquisition of detailed kinetic information that would otherwise be hidden in the averaging over an ensemble of molecules. In this Perspective, we discuss how such approaches have successfully been applied to in vitro-reconstituted systems of increasing complexity.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: Implications for human prolidase

Publisher: Springer Science and Business Media LLC

Date: 10-1998

DOI: 10.1007/S007750050257

Cell-free synthesis and combinatorial selective 15N-labeling of the cytotoxic protein amoebapore A from Entamoeba histolytica

Publisher: Elsevier BV

Date: 11-2009

DOI: 10.1016/J.PEP.2009.06.017

Abstract: Amoebapore A is a pore-forming protein produced by the pathogenic parasite Entamoeba histolytica, which causes human amoebic dysentery. The pore-forming activity of amoebapore A is regulated by pH-dependent dimerization, a prerequisite for membrane insertion and pore formation. Understanding of these important processes has been h ered by the cytotoxicity of amoebapore A, which prevents the production of this protein in cell-based expression systems. In this study, a protocol for the cell-free production of active recombinant amoebapore A is presented. Protein yields of approximately 500 microg/ml of cell-free reaction were achieved. Recombinant amoebapore A was purified using a three-step procedure. To facilitate the structural characterization of the dimeric and pore forms, we adapted the cell-free system to isotope label amoebapore A for NMR studies. The preliminary assignment of a 2D 1H-15N HSQC spectrum of a uniformly 13C/15N-labeled s le was achieved using a combinatorial selective 15N-labeling approach coupled with available 1H(N) chemical shift data, resulting in the unambiguous assignment of resonances from 55 of the 77 residues. To confirm these results and obtain the full sequence-specific assignments of the 2D 1H-15N HSQC spectrum, a 3D HNCA spectrum was recorded. These assignment data will be used to aid the characterization of amoebapore A dimer formation and membrane insertion.

Flexibility Revealed by the 1.85 Angstrom Crystal Structure of the Beta Sliding-Clamp Subunit of Escherichia Coli Dna Polymerase Iii

Publisher: International Union of Crystallography (IUCr)

Date: 27-06-2003

DOI: 10.1107/S0907444903009958

Abstract: The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding cl that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 A [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 A at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 A resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.

In Vitro Plasmid DNA Cleavage by Chromium(V) and -(IV) 2-Hydroxycarboxylato Complexes

Publisher: American Chemical Society (ACS)

Date: 25-03-1999

DOI: 10.1021/TX980229G

Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

Publisher: Elsevier BV

Date: 03-2005

DOI: 10.1074/JBC.M412645200

Mechanism of transcription modulation by the transcription-repair coupling factor

Publisher: Oxford University Press (OUP)

Date: 31-05-2022

DOI: 10.1093/NAR/GKAC449

Abstract: Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

Discovery Projects - Grant ID: DP0663293

Start Date: 2006

End Date: 12-2008

Amount: $365,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0214135

Start Date: 2001

End Date: 12-2002

Amount: $492,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0208428

Start Date: 2002

End Date: 12-2004

Amount: $180,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0342435

Start Date: 2003

End Date: 06-2006

Amount: $435,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100135

Start Date: 08-2010

End Date: 12-2011

Amount: $700,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0877658

Start Date: 05-2008

End Date: 12-2013

Amount: $1,098,934.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180100858

Start Date: 2018

End Date: 12-2020

Amount: $524,298.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180100805

Start Date: 2018

End Date: 02-2021

Amount: $534,573.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP210100167

Start Date: 2021

End Date: 04-2024

Amount: $600,364.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP150100956

Start Date: 2015

End Date: 12-2017

Amount: $778,791.00

Funder: Australian Research Council

Linkage - International - Grant ID: LX0882706

Start Date: 2008

End Date: 12-2012

Amount: $37,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882295

Start Date: 2008

End Date: 06-2009

Amount: $225,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100047

Start Date: 2016

End Date: 09-2016

Amount: $380,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP210100365

Start Date: 02-2021

End Date: 02-2024

Amount: $692,195.00

Funder: Australian Research Council

Linkage - International - Grant ID: LX0668812

Start Date: 06-2006

End Date: 12-2008

Amount: $20,400.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0560751

Start Date: 2005

End Date: 12-2005

Amount: $432,474.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100087

Start Date: 2010

End Date: 12-2010

Amount: $450,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0453832

Start Date: 2004

End Date: 12-2005

Amount: $550,910.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354751

Start Date: 2004

End Date: 12-2004

Amount: $10,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100127

Start Date: 2016

End Date: 12-2016

Amount: $355,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0984797

Start Date: 03-2009

End Date: 12-2014

Amount: $890,000.00

Funder: Australian Research Council

Linkage - CSIRO - Grant ID: LC0348576

Start Date: 10-2003

End Date: 10-2006

Amount: $193,035.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0882009

Start Date: 03-2008

End Date: 09-2011

Amount: $76,881.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989077

Start Date: 2009

End Date: 12-2009

Amount: $225,600.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100085

Start Date: 2011

End Date: 04-2012

Amount: $450,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE140100118

Start Date: 2014

End Date: 12-2014

Amount: $370,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100045

Start Date: 01-2012

End Date: 12-2012

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE190100165

Start Date: 2019

End Date: 12-2019

Amount: $407,609.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989920

Start Date: 2009

End Date: 12-2009

Amount: $750,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100026

Start Date: 04-2018

End Date: 12-2020

Amount: $178,839.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0558652

Start Date: 2005

End Date: 06-2008

Amount: $420,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100078

Start Date: 02-2011

End Date: 12-2011

Amount: $500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100023

Start Date: 03-2017

End Date: 06-2018

Amount: $650,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0454089

Start Date: 03-2004

End Date: 12-2005

Amount: $124,000.00

Funder: Australian Research Council