Jill Trewhella

Solution Scattering Reveals Large Differences in the Global Structures of Type II Protein Kinase A Isoforms

Publisher: Elsevier BV

Date: 03-2006

DOI: 10.1016/J.JMB.2006.01.006

Abstract: Isoform ersity within the protein kinase A (PKA) family is achieved by catalytic (C) subunits binding to different isoforms of regulatory subunit homodimers (R2). In a previous small-angle X-ray scattering study, we showed that the type Ialpha R2 homodimer has a distinctive Y-shaped structure, while the IIalpha and IIbeta homodimers are highly flexible and extended in solution. Here we present the results of X-ray scattering experiments on different isoforms of the PKA holoenzyme (R2C2) and show that the type IIbeta R2 homodimer undergoes a dramatic compaction upon binding C subunits that involves a 10A reduction in radius of gyration (from 56 to 46 A) and a 35 A shortening of the maximum linear dimension (from 180-145 A). In contrast, the type IIalpha R2 homodimer shows very little change in these structural parameters and remains extended upon C-subunit binding. This large difference is surprising given the highly conserved sequence and domain organization for the different R isoforms. A mutant RIIbeta holoenzyme and an RIIalpha/RIIbeta chimera were used to explore the role of the sequence linking different functional domains within RIIbeta in the observed C subunit-induced compaction. Structural modeling was used to aid in interpreting the scattering results in terms of the role of inter-domain and inter-subunit contacts in determining the global conformations of the different isoforms. The results provide an important structural foundation for understanding isoform-specific PKA localization and signaling.

Differential Effects of Substrate on Type I and Type II PKA Holoenzyme Dissociation

Publisher: American Chemical Society (ACS)

Date: 22-04-2004

DOI: 10.1021/BI0499157

Abstract: It has been widely accepted that cAMP activates the protein kinase A (PKA) holoenzyme by dissociating the regulatory and catalytic subunits, thus freeing the catalytic subunit to phosphorylate its targets. However, recent experiments suggest that cAMP does not fully dissociate the holoenzyme. Here, we investigate this mechanism further by using small-angle X-ray scattering to study, at physiological enzyme concentrations, the type Ialpha and type IIbeta holoenzyme structures under equilibrium solution conditions without any labeling of the protein subunits. We observe that while the addition of a molar excess of cAMP to the type Ialpha PKA holoenzyme causes partial dissociation, it is only upon addition of a PKA peptide substrate together with cAMP that full dissociation occurs. Similarly, addition of excess cAMP to the type IIbeta holoenzyme causes only a partial dissociation. However, while the addition of peptide substrate as well as excess cAMP causes somewhat more dissociation, a significant percentage of intact type IIbeta holoenzyme remains. These results confirm that both the type Ialpha and the type IIbeta holoenzymes are more stable in the presence of cAMP than previously thought. They also demonstrate that substrate plays a differential role in the activation of type I versus type II holoenzymes, which could explain some important functional differences between PKA isoforms. On the basis of these data and other recently published data, we propose a structural model of type I holoenzyme activation by cAMP.

Effects of Macromolecular Crowding on an Intrinsically Disordered Protein Characterized by Small-Angle Neutron Scattering with Contrast Matching

Publisher: Elsevier BV

Date: 02-2011

DOI: 10.1016/J.BPJ.2011.01.020

Correction

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.BPJ.2013.08.014

Molecular basis for proton-Dependent anion binding by soybean leghaemoglobin a [28]

Publisher: Springer Science and Business Media LLC

Date: 07-1979

DOI: 10.1038/280087A0

Effects of chain length on oligopeptide hydrogelation

Publisher: Royal Society of Chemistry (RSC)

Date: 2011

DOI: 10.1039/C0SM00919A

A round-robin approach provides a detailed assessment of biomolecular small-angle scattering data reproducibility and yields consensus curves for benchmarking

Publisher: International Union of Crystallography (IUCr)

Date: 20-10-2022

DOI: 10.1107/S2059798322009184

Abstract: Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0–1 Å −1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q -range to .5 Å −1 , but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H 2 O and in D 2 O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q 2.2 Å −1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating s le heterogeneity, but with unavoidable s le dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.

Scattering Intensity Distribution

Publisher: Wiley

Date: 15-01-2002

DOI: 10.1002/047120918X.EMB1378

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity

Publisher: Oxford University Press (OUP)

Date: 31-01-2013

DOI: 10.1093/BRAIN/AWS348

Abstract: Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.

There Is Communication between All Four Ca²⁺-Bindings Sites of Calcineurin B

Publisher: American Chemical Society (ACS)

Date: 15-09-2001

DOI: 10.1021/BI0025060

Abstract: We have used site-directed mutagenesis, flow dialysis, and Fourier transform infrared (FTIR) spectroscopy to study Ca(2+)-binding to the regulatory component of calcineurin. Single Glu-Gln(E --> Q) mutations were used to inactivate each of the four Ca(2+)-binding sites of CnB in turn, generating mutants Q1, Q2, Q3, and Q4, with the number indicating which Ca(2+) site is inactivated. The binding data derived from flow dialysis reveal two pairs of sites in the wild-type protein, one pair with very high affinity and the other with lower affinity Ca(2+)-binding sites. Also, only three sites are titratable in the wild-type protein because one site cannot be decalcified. Mutation of site 2 leaves the protein with only two titratable sites, while mutation of sites 1, 3, or 4 leave three titratable sites that are mostly filled with 3 Ca(2+) equiv added. The binding data further show that each of the single-site mutations Q2, Q3, and Q4 affects the affinities of at least one of the remaining sites. Mutation in either of sites 3 or 4 results in a protein with no high-affinity sites, indicating communication between the two high-affinity sites, most likely sites 3 and 4. Mutation in site 2 decreases the affinity of all three remaining sites, though still leaving two relatively high-affinity sites. The FTIR data support the conclusions from the binding data with respect to the number of titratable sites as well as the impact of each mutation on the affinities of the remaining sites. We conclude therefore that there is communication between all four Ca(2+)-binding sites. In addition, the Ca(2+) induced changes in the FTIR spectra for the wild-type and Q4 mutant are most similar, suggesting that the same three Ca(2+)-binding sites are being titrated, i.e., site 4 is the very high-affinity site under the conditions of the FTIR experiments.

Conformational analysis of PKI(5–22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase

Publisher: Portland Press Ltd.

Date: 12-1989

DOI: 10.1042/BJ2640371

Abstract: Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.

Solution structure of the cAMP-dependent protein kinase

Publisher: Office of Scientific and Technical Information (OSTI)

Date: 31-12-1998

DOI: 10.2172/560750

Light Scattering

Publisher: Wiley

Date: 15-01-2002

DOI: 10.1002/047120918X.EMB0845

Report of the wwPDB Small-Angle Scattering Task Force: Data Requirements for Biomolecular Modeling and the PDB

Publisher: Elsevier BV

Date: 06-2013

DOI: 10.1016/J.STR.2013.04.020

Abstract: This report presents the conclusions of the July 12-13, 2012 meeting of the Small-Angle Scattering Task Force of the worldwide Protein Data Bank (wwPDB Berman et al., 2003) at Rutgers University in New Brunswick, New Jersey. The task force includes experts in small-angle scattering (SAS), crystallography, data archiving, and molecular modeling who met to consider questions regarding the contributions of SAS to modern structural biology. Recognizing there is a rapidly growing community of structural biology researchers acquiring and interpreting SAS data in terms of increasingly sophisticated molecular models, the task force recommends that (1) a global repository is needed that holds standard format X-ray and neutron SAS data that is searchable and freely accessible for download (2) a standard dictionary is required for definitions of terms for data collection and for managing the SAS data repository (3) options should be provided for including in the repository SAS-derived shape and atomistic models based on rigid-body refinement against SAS data along with specific information regarding the uniqueness and uncertainty of the model, and the protocol used to obtain it (4) criteria need to be agreed upon for assessment of the quality of deposited SAS data and the accuracy of SAS-derived models, and the extent to which a given model fits the SAS data (5) with the increasing ersity of structural biology data and models being generated, archiving options for models derived from erse data will be required and (6) thought leaders from the various structural biology disciplines should jointly define what to archive in the PDB and what complementary archives might be needed, taking into account both scientific needs and funding.

Structural determination of larger proteins using stable isotope labeling and NMR spectroscopy

Publisher: Office of Scientific and Technical Information (OSTI)

Date: 04-1996

DOI: 10.2172/212499

A picornaviral loop-to-loop replication complex

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1016/J.JSB.2009.02.010

LIM Domain binding proteins 1 and 2 have different oligomeric states

Publisher: Elsevier BV

Date: 05-2010

DOI: 10.1016/J.JMB.2010.04.006

Abstract: LIM domain binding (Ldb) proteins are important regulators of LIM homeodomain and LIM-only proteins that specify cell fate in many different tissues. An essential feature of these proteins is the ability to self-associate, but there have been no studies that characterise the nature of this self-association. We have used deletion mutagenesis with yeast two-hybrid analysis to define the minimal self-association domains of Ldb1 and Ldb2 as residues 14-200 and 21-197, respectively. We then used a range of different biophysical methods, including sedimentation equilibrium and small-angle X-ray scattering to show that Ldb1(14-200) forms a trimer and Ldb2(21-197) undergoes a monomer-tetramer-octamer equilibrium, where the association in each case is of moderate affinity ( approximately 10(5) M(-1)). These modes of association represent a clear physical difference between these two proteins that otherwise appear to have very similar properties. The levels of association are more complex than previously assumed and emphasise roles of avidity and DNA looping in transcriptional regulation by Ldb1/LIM protein complexes. The abilities of Ldb1 and Ldb2 to form trimers and higher oligomers, respectively, should be considered in models of transcriptional regulation by Ldb1-containing complexes in a wide range of biological processes.

Binding of N-Terminus Fragments of Cardiac Myosin-Binding C-protein to Actin

Publisher: Elsevier BV

Date: 2010

DOI: 10.1016/J.BPJ.2009.12.845

Small-angle scattering for structural biology - Expanding the frontier while avoiding the pitfalls

Publisher: Wiley

Date: 24-03-2010

DOI: 10.1002/PRO.351

Myosin light chain kinase: functional domains and structural motifs

Publisher: Wiley

Date: 12-1998

DOI: 10.1111/J.1365-201X.1998.TB10699.X

Abstract: Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca2+/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca2+/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca2+/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca2+/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca2+/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.

Naysaying the Neutron Scattering Society

Publisher: AIP Publishing

Date: 12-1992

DOI: 10.1063/1.2809905

Solution structure of the bisphosphorylated cardiac specific N-extension of cardiac troponin I

Publisher: Worldwide Protein Data Bank

Date: 11-12-2007

DOI: 10.2210/PDB2JPW/PDB

COAGULATION FACTOR, NMR, 15 STRUCTURES

Publisher: Worldwide Protein Data Bank

Date: 15-05-1997

DOI: 10.2210/PDB1WHF/PDB

Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

Publisher: International Union of Crystallography (IUCr)

Date: 15-05-2009

DOI: 10.1107/S090744490901169X

Dynamic Light Scattering

Publisher: Wiley

Date: 15-01-2002

DOI: 10.1002/047120918X.EMB0440

Spin state equilibria in soybean ferric leghemoglobin and its complexes with formate and acetate

Publisher: Elsevier BV

Date: 05-1979

DOI: 10.1016/0006-291X(79)92106-5

An X-ray crystallographic study of α-d-allopyranosyl α-d-allopyranoside · CaCl2 · 5H2O (a pentadentate complex)

Publisher: Elsevier BV

Date: 02-1978

DOI: 10.1016/S0008-6215(78)80029-9

HIV-1 Reverse Transcriptase Monomers Adopt Multiple Conformations in Solution

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.BPJ.2011.11.284

Growing a thriving international community for small-angle scattering through collaboration

Publisher: International Union of Crystallography (IUCr)

Date: 29-07-2021

DOI: 10.1107/S1600576721007561

Abstract: Small-angle scattering emerged as a tool for studying noncrystalline structures from early observations around 1930 that there was a relationship between the extent of the scattering and the size of the scattering object. André Guinier, a leading figure in the development of the field, noted in his summary findings from the first Conference on Small Angle Scattering in 1958 that the technique would be of value to study `submicroscopical inhomogeneities' and further provided a means of `observation [that had] in the past restricted the field of application of the X-ray method.' In 1965 the first of what became a highly successful series of Small-Angle Scattering (SAS) meetings held approximately every three years took place in Syracuse, NY, USA, and many of these ongoing meetings published their proceedings and highlights in the International Union of Crystallography (IUCr) Journal of Applied Crystallography . Since the early 2000s, the relationship between the international SAS community represented at the triennial SAS meetings and the IUCr has been strengthened and deepened through formal cooperation and collaboration in a number of mutually beneficial activities that have supported the growth and health of the field and the IUCr.

Solution structure studies of monomeric human TIP47/perilipin‐3 reveal a highly extended conformation

Publisher: Wiley

Date: 25-05-2012

DOI: 10.1002/PROT.24095

The N-Terminal Domains of Myosin Binding Protein C Can Bind Polymorphically to F-Actin

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.JMB.2011.07.056

Contrast Variation

Publisher: Wiley

Date: 15-01-2002

DOI: 10.1002/047120918X.EMB0310

Recent advances in small-angle scattering and its expanding impact in structural biology

Publisher: Elsevier BV

Date: 2022

DOI: 10.1016/J.STR.2021.09.008

Abstract: Applications of small-angle scattering (SAS) in structural biology have benefited from continuing developments in instrumentation, tools for data analysis, modeling capabilities, standards for data and model presentation, and data archiving. The interplay of these capabilities has enabled SAS to contribute to advances in structural biology as the field pushes the boundaries in studies of biomolecular complexes and assemblies as large as whole cells, membrane proteins in lipid environments, and dynamic systems on time scales ranging from femtoseconds to hours. This review covers some of the important advances in biomolecular SAS capabilities for structural biology focused on over the last 5 years and presents highlights of recent applications that demonstrate how the technique is exploring new territories.

An optimized SEC-SAXS system enabling high X-ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis

Publisher: International Union of Crystallography (IUCr)

Date: 02-2018

DOI: 10.1107/S1600576717017101

Abstract: A new optimized size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) system for biomolecular SAXS at the Australian Synchrotron SAXS/WAXS beamline has been developed. The compact configuration reduces s le dilution to maximize sensitivity. Coflow s le presentation allows an 11-fold increase in flux on s le without capillary fouling, improving throughput and data quality, which are now primarily limited by the full flux available on the beamline. Multi-wavelength fibre optic UV analysis in close proximity to the X-ray beam allows for accurate concentration determination for s les with known UV extinction coefficients and thus estimation of the molecular weight of the scattering particle from the forward X-ray scattering intensity. Fast-flow low-volume SEC columns provide s le throughput competitive with batch concentration series measurements, albeit with a concomitant reduction of potential resolution relative to lower flow rates and larger SEC columns. The performance of the system is demonstrated using a set of model proteins, and its utility to solve various challenges is illustrated with a erse suite of protein s les. These developments increase the quality and rigor of SEC-SAXS analysis and open new avenues for biomolecular solution SEC-SAXS studies that have been challenged by low s le yields, temporal instability, radiation sensitivity and complex mixtures.

The Relative Orientation of Gla and EGF Domains in Coagulation Factor X Is Altered by Ca²⁺ Binding to the First EGF Domain. A Combined NMR−Small Angle X-ray Scattering Study^,

Publisher: American Chemical Society (ACS)

Date: 1996

DOI: 10.1021/BI960633J

The Solution Structure of the Sac7d/DNA Complex:  A Small-Angle X-ray Scattering Study

Publisher: American Chemical Society (ACS)

Date: 23-07-1999

DOI: 10.1021/BI990782C

Book reviews

Publisher: Informa UK Limited

Date: 12-2010

DOI: 10.1080/07294360.2010.514699

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis

Publisher: Springer Science and Business Media LLC

Date: 10-1985

DOI: 10.1007/BF01871088

Abstract: Core-shell microgels containing sensors/dyes in a matrix were fabricated by two-stage free radical precipitation polymerization method for ratiometric sensing/imaging. The microgels composing of poly(N-isopropylacrylamide) (PNIPAm) shell exhibits a low critical solution temperature (LCST), underwent an entropically driven transition from a swollen state to a deswollen state, which exhibit a hydrodynamic radius of ∼ 450 nm at 25 °C (in vitro) and ∼ 190 nm at 37 °C (in vivo). The microgel's ability of escaping from lysosome into cytosol makes the microgel be a potential candidate for cytosolic delivery of sensors robes. Non-invasive imaging/sensing in Antigen-presenting cells (APCs) was feasible by monitoring the changes of fluorescence intensity ratios. Thus, these biocompatible microgels-based imaging/sensing agents may be expected to expand current molecular imaging/sensing techniques into methods applicable to studies in vivo, which could further drive APC-based treatments.

Small-Angle X-ray Scattering Reveals the N-Terminal Domain Organization of Cardiac Myosin Binding Protein C

Publisher: Elsevier BV

Date: 04-2008

DOI: 10.1016/J.JMB.2008.01.080

Abstract: Myosin binding protein C (MyBP-C) is a multidomain accessory protein of striated muscle sarcomeres. Three domains at the N-terminus of MyBP-C (C1-m-C2) play a crucial role in maintaining and modulating actomyosin interactions. The cardiac isoform has an additional N-terminal domain (C0) that is postulated to provide a greater level of regulatory control in cardiac muscle. We have used small-angle X-ray scattering, ab initio shape restoration, and rigid-body modeling to determine the average shape and spatial arrangement of the four N-terminal domains of cardiac MyBP-C (C0C2) and a three-domain variant that is analogous to the N-terminus of the skeletal isoform (C1C2). We found that the domains of both proteins are tandemly arranged in a highly extended configuration that is sufficiently long to span the interfilament cross-bridge distances in vivo and, hence, be poised to modulate these interactions. The average spatial organization of the C1, m, and C2 domains is not significantly perturbed by the removal of the cardiac-specific C0 domain, suggesting that the interdomain interfaces, while relatively small in area, have a degree of rigidity. Modeling the C0C2 and C1C2 scattering data reveals that the structures of the C0 and m domains (also referred to as the 'MyBP motif') are compact and have dimensions that are consistent with the immunoglobulin fold superfamily of proteins. Sequence analysis, homology modeling, and circular dichroism experiments support the conclusion that the previously undetermined structures of these domains can be characterized as having an immunoglobulin-like fold. Atomic models using the known NMR structures for C1 and C2 as well as homology models for the C0 and m domains provide insights into the placement of conserved serine residues of the m domain that are phosphorylated in vivo and cause a change in muscle fiber contraction by abolishing interactions with myosin.

Growing a thriving international community for small-angle scattering through collaboration. Corrigendum

Publisher: International Union of Crystallography (IUCr)

Date: 16-11-2021

DOI: 10.1107/S160057672101164X

Abstract: Errors in the article by Jill Trewhella [ J. Appl. Cryst . (2021), 54 , 1029–1033] are corrected.

Solution Structure of Heavy Meromyosin by Small-angle Scattering

Publisher: Elsevier BV

Date: 02-2003

DOI: 10.1074/JBC.M210558200

Conformationally constrained analogs of protein kinase inhibitor(6–22)amide: Effect of turn structures in the center of the peptide on inhibition of cAMP‐dependent protein kinase

Publisher: Wiley

Date: 03-1995

DOI: 10.1002/PRO.5560040307

Abstract: The high-affinity interaction between protein kinase inhibitor (PKI)(6-22)amide(Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly- Arg-Arg-Asn- Ala-Ile22-NH2) and the catalytic subunit of cAMP-dependent protein kinase requires both the N-terminal Thr6 to Ile11 sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg15 to Ile22. Small angle X-ray scattering data indicate that PKI(6-22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem J 264:371-380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a beta-turn structure might be located in the -Ala12-Ser-Gly-Arg15-connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry 26:7641-7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6-22)amide were synthesized and used to study the structure-function relationships of this central portion of the inhibitor. (Des12-14)PKI(6-22) amide exhibited over a 200-fold loss in inhibitory activity. Replacement of the omitted -Ala12-Ser-Gly14-sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The D-alanine14 PKI analog was as potent as the parent peptide, whereas the beta-alanine14 and the sarcosine14 analogs were only 10-fold less active. Several peptides that promoted a beta-turn structure at residues 12-15 showed about 200-fold decreases in inhibitory activity. Two constrained analogs that could not assume a beta-turn conformation were only 30-fold less potent than PKI(6-22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12-15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal beta-turn at this position is not required and is actually detrimental for a high-affinity interaction of PKI(6-22)amide with the enzyme. These results are interpreted in light of the Fourier-transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science 253:414-420).

Potential biophysical application of the Los Alamos infrared free electron laser: DNA spectroscopy

Publisher: Elsevier BV

Date: 10-1990

DOI: 10.1016/0168-9002(90)91311-X

The structure of TTHA0988 from Thermus thermophilus, a KipI-KipA homologue incorrectly annotated as an allophanate hydrolase

Publisher: International Union of Crystallography (IUCr)

Date: 08-01-2011

DOI: 10.1107/S0907444910051127

Small‐Angle Scattering

Publisher: Wiley

Date: 15-01-2002

DOI: 10.1002/047120918X.EMB1440

X ‐Ray Scattering

Publisher: Wiley

Date: 15-01-2002

DOI: 10.1002/047120918X.EMB1675

The Solution Structure of a Cardiac Troponin C−Troponin I−Troponin T Complex Shows a Somewhat Compact Troponin C Interacting with an Extended Troponin I−Troponin T Component

Publisher: American Chemical Society (ACS)

Date: 12-2002

DOI: 10.1021/BI026687C

Abstract: We have investigated the structure of the cTnC-cTnI-cTnT(198-298) calcium-saturated, ternary cardiac troponin complex by small-angle scattering with contrast variation. Shape restoration was also applied to the scattering information resulting from the deuterated cTnC subunit, the unlabeled cTnI-cTnT(198-298) subunits, and the entire complex. The experimental results and modeling indicate that cTnC adopts a partially collapsed conformation, while the cTnI-cTnT(198-298) components have an extended, rod-like structure. Shape restoration applied to the X-ray scattering data and the entire contrast variation series suggest that cTnC and the cTnI-cTnT(198-298) component lie with their long axes roughly parallel to one another with a relatively small surface area for interaction. Our findings indicate that the nature of the interactions between TnC and the TnI-TnT component differs significantly between the cardiac and skeletal isoforms as evidenced by the different degrees of compactness between the cardiac TnC and skeletal TnC in their respective ternary complexes and the fact that the cTnC subunit is not highly intertwined with the other subunits, as observed in the binary complex of the skeletal isoforms [Olah, G. A., and Trewhella, J. (1994) Biochemistry 33, 12800-12806].

Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase

Publisher: Proceedings of the National Academy of Sciences

Date: 18-02-2003

DOI: 10.1073/PNAS.0534892100

Abstract: Using small-angle x-ray scattering, we have observed the cGMP-induced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (Δ 1–52 PKG-Iβ). On saturation with cGMP, the radius of gyration of Δ 1–52 PKG-Iβ increases from 29.4 ± 0.1 Å to 40.1 ± 0.7 Å, and the maximum linear dimension increases from 90 Å ± 10% to 130 Å ± 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to Δ 1–52 PKG-Iβ indicates that elongation of Δ 1–52 PKG-Iβ requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of Δ 1–52 PKG-Iβ. We also predict that there is little or no cooperativity in cGMP binding to the two sites of Δ 1–52 PKG-Iβ under the conditions used here. Results obtained by using the Δ 1–52 PKG-Iβ monomer indicate that a previously observed elongation of PKG-Iα is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-Iβ activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.

Development of experimental techniques to study protein and nucleic acid structures

Publisher: Office of Scientific and Technical Information (OSTI)

Date: 04-1996

DOI: 10.2172/212551

The Solution Structure of the DNA Double-Stranded Break Repair Protein Ku and Its Complex with DNA:  A Neutron Contrast Variation Study

Publisher: American Chemical Society (ACS)

Date: 30-01-1999

DOI: 10.1021/BI9825246

The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity

Publisher: Elsevier BV

Date: 12-1998

DOI: 10.1006/JMBI.1998.2028

Abstract: Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite ersified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct ( approximately 15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.

Ligand-induced Conformational Changes and Conformational Dynamics in the Solution Structure of the Lactose Repressor Protein

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1016/J.JMB.2007.11.067

Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation

Publisher: Elsevier BV

Date: 11-1998

DOI: 10.1074/JBC.273.46.30448

Characterization of the solution structure of a neuroligin/β-neurexin complex

Publisher: Elsevier BV

Date: 09-2008

DOI: 10.1016/J.CBI.2008.04.040

Radius Of Gyration

Publisher: Wiley

Date: 15-01-2002

DOI: 10.1002/047120918X.EMB1272

Orienting rigid and flexible biological assemblies in ferrofluids for small-angle neutron scattering studies

Publisher: Elsevier BV

Date: 11-1991

DOI: 10.1016/S0006-3495(91)82153-1

A Day in the World Science Alliance

Publisher: American Association for the Advancement of Science (AAAS)

Date: 10-12-1999

DOI: 10.1126/SCIENCE.286.5447.2104

Denatured states of ribonuclease A have compact dimensions and residual secondary structure

Publisher: American Chemical Society (ACS)

Date: 09-1992

DOI: 10.1021/BI00150A029

Abstract: Using small-angle X-ray scattering and Fourier transform infrared spectroscopy, we have determined that the thermally denatured state of native ribonuclease A is on average a compact structure having residual secondary structure. Under strongly reducing conditions, the protein further unfolds into a looser structure with larger dimensions but still retains a comparable amount of secondary structure. The dimensions of the thermally and chemically denatured states of the reduced protein are different but both are more compact than is predicted for a random coil of the same length. These results demonstrate that thermal denaturation in ribonuclease A is not a simple two-state transition from a native to a completely disordered random coil state.

Neutron diffraction studies of bacteriorhodopsin

Publisher: Elsevier BV

Date: 1986

DOI: 10.1016/S0378-4363(86)80067-5

News Reports

Publisher: Informa UK Limited

Date: 1992

DOI: 10.1080/10448639208218761

A Structural Model of the Catalytic Subunit-regulatory Subunit Dimeric Complex of the cAMP-dependent Protein Kinase

Publisher: Elsevier BV

Date: 04-2002

DOI: 10.1074/JBC.M110298200

Fractal dimension of an intrinsically disordered protein: Small‐angle X‐ray scattering and computational study of the bacteriophage λ N protein

Publisher: Wiley

Date: 26-10-2011

DOI: 10.1002/PRO.739

Structural basis for partial redundancy in a class of transcription factors, the LIM homeodomain proteins, in neural cell type specification.

Publisher: Elsevier BV

Date: 12-2011

DOI: 10.1074/JBC.M111.248559

Calmodulin Remains Extended upon Binding to Smooth Muscle Caldesmon:  A Combined Small-Angle Scattering and Fourier Transform Infrared Spectroscopy Study

Publisher: American Chemical Society (ACS)

Date: 18-03-2000

DOI: 10.1021/BI992638X

Abstract: We show that calmodulin (CaM) has an extended conformation in its complexes with sequences from the smooth muscle thin filament protein caldesmon (CaD) by using small-angle X-ray and neutron scattering with contrast variation. The CaD sequences used in these experiments were a C-terminal fragment, 22kCaD, and a smaller peptide sequence within this fragment, MG56C. Each of these sequences contains the CaM-binding sites A and B previously shown to interact with the C- and N-terminal lobes of CaM, respectively [Wang et al. (1997) Biochemistry 36, 15026]. By modeling the scattering data, we show that the majority of the MG56C sequence binds to the N-terminal domain of CaM. FTIR data on CaM complexed with 22kCaD or with MG56C peptide show the 22kCaD sequence contains unordered, helix, and extended structures, and that the extended structures reside primarily in the MG56C portion of the sequence. There are small changes in secondary structure, involving approximately 12 residues, induced by CaM binding to CaD. These changes involve a net decrease in extended structures accompanied by an increase in alpha-helix, and they occur within the CaM and/or in the MG56C sequence.

Neutrons reveal how nature uses structural themes and variation in biological regulation

Publisher: Elsevier BV

Date: 11-2006

DOI: 10.1016/J.PHYSB.2006.05.118

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

Publisher: Wiley

Date: 25-03-2011

DOI: 10.1002/BIP.21624

Abstract: Small-angle X-ray and neutron scattering with contrast variation have made important contributions in advancing our understanding of muscle regulatory protein structures in the context of the dynamic molecular processes governing muscle action. The contributions of the scattering investigations have depended upon the results of key crystallographic, NMR, and electron microscopy experiments that have provided detailed structural information that has aided in the interpretation of the scattering data. This review will cover the advances made using small-angle scattering techniques, in combination with the results from these complementary techniques, in probing the structures of troponin and myosin binding protein C. A focus of the troponin work has been to understand the isoform differences between the skeletal and cardiac isoforms of this major calcium receptor in muscle. In the case of myosin binding protein C, significant data are accumulating, indicating that this protein may act to modulate the primary calcium signals from troponin, and interest in its biological role has grown because of linkages between gene mutations in the cardiac isoform and serious heart disease.

Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

Publisher: Proceedings of the National Academy of Sciences

Date: 25-11-2008

DOI: 10.1073/PNAS.0808903105

Abstract: Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin.

Erratum to Domain Organization of the Monomeric Form of the Tom70 Mitochondrial Import Receptor [J. Mol. Biol. 388 (2009) 1043-1058] (DOI

Publisher: Elsevier BV

Date: 07-2009

DOI: 10.1016/J.JMB.2009.05.020

Calmodulin Binds a Highly Extended HIV-1 MA Protein That Refolds Upon Its Release

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1016/J.BPJ.2012.06.042

The solution structures of calmodulin and its complexes with synthetic peptides based on target enzyme binding domains

Publisher: Elsevier BV

Date: 06-1992

DOI: 10.1016/0143-4160(92)90051-S

Abstract: Small-angle X-ray and neutron scattering experiments have given important information on the solution structures of calmodulin and its complexes with synthetic peptides used to model target enzyme interactions. In combination with crystallographic data, site directed mutagenesis and various spectroscopic studies, these experiments have contributed to our understanding of the solution structure of calmodulin in different functional states. We have gained important insights into the conformational flexibility in calmodulin that appears to be crucial to its regulatory functions. Specifically, flexibility in the interconnecting helix region of calmodulin has been shown to play a critical role in facilitating calmodulin's binding to a wide variety of target enzymes whose activities are thus regulated. This review will focus mainly on the contributions small-angle scattering has made to our understanding of the solution structure of calmodulin in the context of other studies, with particular regard to circular dichroism and Fourier transform infrared studies that complement the small-angle scattering data.

A model of troponin‐I in complex with troponin‐C using hybrid experimental data: The inhibitory region is a β‐hairpin

Publisher: Wiley

Date: 2000

DOI: 10.1110/PS.9.7.1312

Abstract: We present a model for the skeletal muscle troponin-C (TnC)/troponin-I (TnI) interaction, a critical molecular switch that is responsible for calcium-dependent regulation of the contractile mechanism. Despite concerted efforts by multiple groups for more than a decade, attempts to crystallize troponin-C in complex with troponin-I, or in the ternary troponin-complex, have not yet delivered a high-resolution structure. Many groups have pursued different experimental strategies, such as X-ray crystallography, NMR, small-angle scattering, chemical cross-linking, and fluorescent resonance energy transfer (FRET) to gain insights into the nature of the TnC/TnI interaction. We have integrated the results of these experiments to develop a model of the TnC/TnI interaction, using an atomic model of TnC as a scaffold. The TnI sequence was fit to each of two alternate neutron scattering envelopes: one that winds about TnC in a left-handed sense (Model L), and another that winds about TnC in a right-handed sense (Model R). Information from crystallography and NMR experiments was used to define segments of the models. Tests show that both models are consistent with available cross-linking and FRET data. The inhibitory region TnI(95-114) is modeled as a flexible beta-hairpin, and in both models it is localized to the same region on the central helix of TnC. The sequence of the inhibitory region is similar to that of a beta-hairpin region of the actin-binding protein profilin. This similarity supports our model and suggests the possibility of using an available profilin/actin crystal structure to model the TnI/actin interaction. We propose that the beta-hairpin is an important structural motif that communicates the Ca2+-activated troponin regulatory signal to actin.

Human Cardiac Myosin Binding Protein C: Structural Flexibility within an Extended Modular Architecture

Publisher: Elsevier BV

Date: 12-2011

DOI: 10.1016/J.JMB.2011.10.029

Abstract: New insights into the modular organization and flexibility of the N-terminal half of human cardiac myosin binding protein C (cMyBP-C) and information on the association state of the full-length protein have been deduced from a combined small-angle X-ray scattering (SAXS) and NMR study. SAXS data show that the first five immunoglobulin domains of cMyBP-C, which include those implicated in interactions with both myosin and actin, remain monodisperse and monomeric in solution and have a highly extended yet distinctively 'bent' modular arrangement that is similar to the giant elastic muscle protein titin. Analyses of the NMR and SAXS data indicate that a proline/alanine-rich linker connecting the cardiac-specific N-terminal C0 domain to the C1 domain provides significant structural flexibility at the N-terminus of the human isoform, while the modular arrangement of domains C1-C2-C3-C4 is relatively fixed. Domain fragments from the C-terminal half of the protein have a propensity to self-associate in vitro, while full-length bacterially expressed cMyBP-C forms flexible extended dimers at micromolar protein concentrations. In summary, our studies reveal that human cMyBP-C combines a distinctive modular architecture with regions of flexibility and that the N-terminal half of the protein is sufficiently extended to span the range of interfilament distances s led within the dynamic environment of heart muscle. These structural features of cMyBP-C could facilitate its putative role as a molecular switch between actin and myosin and may contribute to modulating the transverse pliancy of the C-zone of the A-band across muscle sarcomeres.

Solution Structure of Ectodomains of the Insulin Receptor Family: The Ectodomain of the Type 1 Insulin-Like Growth Factor Receptor Displays Asymmetry of Ligand Binding Accompanied by Limited Conformat

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1016/J.JMB.2009.10.011

Abstract: The insulin receptor (IR) and the homologous Type 1 insulin-like growth factor receptor (IGF-1R) are cell-surface tyrosine kinase receptors that effect signaling within the respective pathways of glucose metabolism and normal human growth. While ligand binding to these receptors is assumed to result in a structural transition within the receptor ectodomain that then effects signal transduction across the cell membrane, little is known about the molecular detail of these events. Presented here are small-angle X-ray scattering data obtained from the IR and IGF-1R ectodomains in solution. We show that, in solution, the ectodomains of IR and IGF-1R have a domain disposition that is very similar to that seen in the crystal structure of the ectodomain of IR, despite the constituent domains being in relatively sparse contact and potentially mobile. We also show that the IGF-1R ectodomain is capable of binding up to three molecules of IGF-1 in solution, with surprisingly little apparent change in relative domain disposition compared to the apo form. While the observed 3:1 ligand-binding stoichiometry appears to contradict earlier explanations of the absence of a bell-shaped dose-response curve for IGF-1R in ligand displacement assays, it is readily understood in the context of the harmonic oscillator model of the negative cooperativity of ligand binding to IGF-1R. Taken together, our findings suggest that the structural movements within these receptors upon ligand binding are small and are possibly limited to local rotation of domains.

Solution structure of the LIM-homeodomain transcription factor complex Lhx3/Ldb1 and the effects of a pituitary mutation on key Lhx3 interactions.

Publisher: Public Library of Science (PLoS)

Date: 25-07-2012

DOI: 10.1371/JOURNAL.PONE.0040719

Phosphorylation-dependent Conformational Transition of the Cardiac Specific N-Extension of Troponin I in Cardiac Troponin

Publisher: Elsevier BV

Date: 10-2007

DOI: 10.1016/J.JMB.2007.08.035

Abstract: We present here the solution structure for the bisphosphorylated form of the cardiac N-extension of troponin I (cTnI(1-32)), a region for which there are no previous high-resolution data. Using this structure, the X-ray crystal structure of the cardiac troponin core, and uniform density models of the troponin components derived from neutron contrast variation data, we built atomic models for troponin that show the conformational transition in cardiac troponin induced by bisphosphorylation. In the absence of phosphorylation, our NMR data and sequence analyses indicate a less structured cardiac N-extension with a propensity for a helical region surrounding the phosphorylation motif, followed by a helical C-terminal region (residues 25-30). In this conformation, TnI(1-32) interacts with the N-lobe of cardiac troponin C (cTnC) and thus is positioned to modulate myofilament Ca2+-sensitivity. Bisphosphorylation at Ser23/24 extends the C-terminal helix (residues 21-30) which results in weakening interactions with the N-lobe of cTnC and a re-positioning of the acidic amino terminus of cTnI(1-32) for favorable interactions with basic regions, likely the inhibitory region of cTnI. An extended poly(L-proline)II helix between residues 11 and 19 serves as the rigid linker that aids in re-positioning the amino terminus of cTnI(1-32) upon bisphosphorylation at Ser23/24. We propose that it is these electrostatic interactions between the acidic amino terminus of cTnI(1-32) and the basic inhibitory region of troponin I that induces a bending of cTnI at the end that interacts with cTnC. This model provides a molecular mechanism for the observed changes in cross-bridge kinetics upon TnI phosphorylation.

Structural basis of interprotein electron transfer in bacterial sulfite oxidation

Publisher: eLife Sciences Publications, Ltd

Date: 19-12-2015

DOI: 10.7554/ELIFE.09066

Abstract: Interprotein electron transfer underpins the essential processes of life and relies on the formation of specific, yet transient protein-protein interactions. In biological systems, the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs), which interact with an electron acceptor for catalytic turnover. Here, we report the structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the SorT/SorU interaction show the complex is dynamic in solution, and that the proteins interact with Kd = 13.5 ± 0.8 μM. The crystal structures of the oxidized SorT and SorU, both in isolation and in complex, reveal the interface to be remarkably electrostatic, with an unusually large number of direct hydrogen bonding interactions. The assembly of the complex is accompanied by an adjustment in the structure of SorU, and conformational s ling provides a mechanism for dissociation of the SorT/SorU assembly.

Ordered macromolecular structures in ferrofluid mixtures

Publisher: American Physical Society (APS)

Date: 03-04-1989

DOI: 10.1103/PHYSREVLETT.62.1667

A chemically modified α-amylase with a molten-globule state has entropically driven enhanced thermal stability†

Publisher: Oxford University Press (OUP)

Date: 09-08-2010

DOI: 10.1093/PROTEIN/GZQ051

Abstract: The thermostability properties of TAA were investigated by chemically modifying carboxyl groups on the surface of the enzyme with AMEs. The TAA(MOD) exhibited a 200% improvement in starch-hydrolyzing productivity at 60 degrees C. By studying the kinetic, thermodynamic and biophysical properties, we found that TAA(MOD) had formed a thermostable, MG state, in which the unfolding of the tertiary structure preceded that of the secondary structure by at least 20 degrees C. The X-ray crystal structure of TAA(MOD) revealed no new permanent interactions (electrostatic or other) resulting from the modification. By deriving thermodynamic activation parameters of TAA(MOD), we rationalised that thermostabilisation have been caused by a decrease in the entropy of the transition state, rather than being enthalpically driven. Far-UV CD shows that the origin of decreased entropy may have arisen from a higher helical content of TAA(MOD). This study provides new insight into the intriguing properties of an MG state resulting from the chemical modification of TAA.

Small-Angle X-ray Scattering of Reduced Ribonuclease A: Effects of Solution Conditions and Comparisons with a Computational Model of Unfolded Proteins

Publisher: Elsevier BV

Date: 04-2008

DOI: 10.1016/J.JMB.2008.02.009

Progressive Cyclic Nucleotide-induced Conformational Changes in the cGMP-dependent Protein Kinase Studied by Small Angle X-ray Scattering in Solution

Publisher: Elsevier BV

Date: 12-1997

DOI: 10.1074/JBC.272.50.31929

Assignment of segments of the bacteriorhodopsin sequence to positions in the structural map

Publisher: Elsevier BV

Date: 06-1983

DOI: 10.1016/S0006-3495(83)84391-4

Abstract: Complete understanding of the neural correlates of cognitive processes requires investigation of both event- and state-related correlates of cognitive performance as well as their interaction. Neuroimaging studies using blocked designs confound these two types of processes and studies using event-related designs focus exclusively on the detection of transient effects. Recent fMRI studies used mixed blocked/event-related designs and found that transient and sustained activity can be dissociated, but it is not yet known how event-related and state-related processing interact. Here we used a phonological categorization paradigm in a mixed blocked/event-related design to investigate where in the brain transient activity interacts with sustained activity. Task difficulty was parametrically manipulated based on in idually determined categorization thresholds. We found an interaction effect of transient and sustained activity in the left precuneus. In this cortical structure transient activity increased with increasing task difficulty, while sustained neural activity decreased with increasing task difficulty. Our data suggest that sustained activity is enhanced during processing of an easy task, presumably because of ongoing internally cued endogenous processing, still allowing effortless processing of transient stimuli. During performance of a difficult task, sustained activity in the precuneus is reduced to provide resources for processing incoming stimuli. Processing of stimuli that are expected to be difficult elicits increased transient responses independent of the actual physical properties of the stimuli. In showing an interaction between transient and sustained activity in the precuneus, the present results accommodate seemingly erging results from previous studies using event-related or blocked designs and expand the knowledge emerging from previous studies using mixed blocked/event-related designs.

Potential Biophysical Application Of The Los Alamos Infrared Free Electron Laser: DNA Spectroscopy

Publisher: IEEE

DOI: 10.1109/FEL.1989.716113

Calmodulin Binding to Myosin Light Chain Kinase Begins at Substoichiometric Ca²⁺ Concentrations:  A Small-Angle Scattering Study of Binding and Conformational Transitions

Publisher: American Chemical Society (ACS)

Date: 12-1998

DOI: 10.1021/BI981656W

Abstract: We have used small-angle scattering to study the calcium dependence of the interactions between calmodulin (CaM) and skeletal muscle myosin light chain kinase (MLCK), as well as the conformations of the complexes that form. Scattering data were measured from equimolar mixtures of a functional MLCK and CaM or a mutated CaM (B12QCaM) incompetent to bind Ca2+ in its N-terminal domain, with increasing Ca2+ concentrations. To evaluate differences between CaM-enzyme versus CaM-peptide interactions, similar Ca2+ titration experiments were performed using synthetic peptides based on the CaM-binding sequence from MLCK (MLCK-I). Our data show there are different determinants for CaM binding the isolated peptide sequence compared to CaM binding to the same sequences within the enzyme. For ex le, binding of either CaM or B12QCaM to the MLCK-I peptide is observed even in the presence of EGTA, whereas binding of CaM to the enzyme requires Ca2+. The peptide studies also show that the conformational collapse of CaM requires both the N and C domains of CaM to be competent for Ca2+ binding as well as interactions with each end of MLCK-I, and it occurs at approximately 2 mol of Ca2+/mol of CaM. We show that CaM binding to the MLCK enzyme begins at substoichiometric concentrations of Ca2+ (< or = 2 mol of Ca2+/mol of CaM), but that the final compact structure of CaM with the enzyme requires saturating Ca2+. In addition, MLCK enzyme does bind to 2Ca2+ x B12QCaM, although this complex is more extended than the complex with native CaM. Our results support the hypothesis that CaM regulation of MLCK involves an initial binding step at less than saturating Ca2+ concentrations and a subsequent activation step at higher Ca2+ concentrations.

MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies

Publisher: International Union of Crystallography (IUCr)

Date: 16-01-2008

DOI: 10.1107/S0021889807055136

Abstract: Small-angle neutron scattering with contrast variation can fill important gaps in our understanding of biomolecular assemblies, providing constraints that can aid in the construction of molecular models and in subsequent model refinements. This paper describes the implementation of simple tools for analysing neutron contrast variation data, accessible via a user-friendly web-based interface (www.mmb.usyd.edu.au/NCVWeb/). There are three modules accessible from the website to analyse neutron contrast variation data from bimolecular complexes. The first module, Contrast , computes neutron contrasts of each component of the complex required by the other two modules the second module, R g , analyses the contrast dependence of the radii of gyration to yield information relating to the size and disposition of each component in the complex and the third, Compost , decomposes the contrast variation series into composite scattering functions, which contain information regarding the shape of each component of the complex, and their orientation with respect to each other.

The Structure of the Muscle Protein Complex 4Ca2+·Troponin C·Troponin I

Publisher: Springer US

Date: 1996

DOI: 10.1007/978-1-4615-5847-7_14

AlphaFold-predicted protein structures and small-angle X-ray scattering: insights from an extended examination of selected data in the Small-Angle Scattering Biological Data Bank

Publisher: International Union of Crystallography (IUCr)

Date: 20-07-2023

DOI: 10.1107/S1600576723005344

Abstract: By providing predicted protein structures from nearly all known protein sequences, the artificial intelligence program AlphaFold (AF) is having a major impact on structural biology. While a stunning accuracy has been achieved for many folding units, predicted unstructured regions and the arrangement of potentially flexible linkers connecting structured domains present challenges. Focusing on single-chain structures without prosthetic groups, an earlier comparison of features derived from small-angle X-ray scattering (SAXS) data taken from the Small-Angle Scattering Biological Data Bank (SASBDB) is extended to those calculated using the corresponding AF-predicted structures. Selected SASBDB entries were carefully examined to ensure that they represented data from monodisperse protein solutions and had sufficient statistical precision and q resolution for reliable structural evaluation. Three ex les were identified where there is clear evidence that the single AF-predicted structure cannot account for the experimental SAXS data. Instead, excellent agreement is found with ensemble models generated by allowing for flexible linkers between high-confidence predicted structured domains. A pool of representative structures was generated using a Monte Carlo method that adjusts backbone dihedral allowed angles along potentially flexible regions. A fast ensemble modelling method was employed that optimizes the fit of pair distance distribution functions [ P ( r ) versus r ] and intensity profiles [ I ( q ) versus q ] computed from the pool to their experimental counterparts. These results highlight the complementarity between AF prediction, solution SAXS and molecular dynamics/conformational s ling for structural modelling of proteins having both structured and flexible regions.

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

Publisher: Springer Science and Business Media LLC

Date: 23-03-2016

DOI: 10.1038/SREP23123

Abstract: In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis , the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis , alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.

Sol and gel states in peptide hydrogels visualized by Gd(III)‐enhanced magnetic resonance imaging

Publisher: Wiley

Date: 2011

DOI: 10.1002/BIP.21612

Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop

Publisher: Elsevier BV

Date: 07-2015

DOI: 10.1016/J.STR.2015.05.013

COAGULATION FACTOR, NMR, 20 STRUCTURES

Publisher: Worldwide Protein Data Bank

Date: 15-05-1997

DOI: 10.2210/PDB1WHE/PDB

Low‐angle Scattering of Neutrons and X‐rays

Publisher: Wiley

Date: 21-08-2001

DOI: 10.1038/NPG.ELS.0003047

Subdividing Repressor Function: DNA Binding Affinity, Selectivity, and Allostery Can Be Altered by Amino Acid Substitution of Nonconserved Residues in a LacI/GalR Homologue

Publisher: American Chemical Society (ACS)

Date: 11-07-2008

DOI: 10.1021/BI800443K

2017 publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution: an update

Publisher: International Union of Crystallography (IUCr)

Date: 18-08-2017

DOI: 10.1107/S2059798317011597

Abstract: In 2012, preliminary guidelines were published addressing s le quality, data acquisition and reduction, presentation of scattering data and validation, and modelling for biomolecular small-angle scattering (SAS) experiments. Biomolecular SAS has since continued to grow and authors have increasingly adopted the preliminary guidelines. In parallel, integrative/hybrid determination of biomolecular structures is a rapidly growing field that is expanding the scope of structural biology. For SAS to contribute maximally to this field, it is essential to ensure open access to the information required for evaluation of the quality of SAS s les and data, as well as the validity of SAS-based structural models. To this end, the preliminary guidelines for data presentation in a publication are reviewed and updated, and the deposition of data and associated models in a public archive is recommended. These guidelines and recommendations have been prepared in consultation with the members of the International Union of Crystallography (IUCr) Small-Angle Scattering and Journals Commissions, the Worldwide Protein Data Bank (wwPDB) Small-Angle Scattering Validation Task Force and additional experts in the field.

Effect of Temperature During Assembly on the Structure and Mechanical Properties of Peptide-Based Materials

Publisher: American Chemical Society (ACS)

Date: 19-05-2010

DOI: 10.1021/BM100138M

The Motif of Human Cardiac Myosin-binding Protein C Is Required for Its Ca2+-dependent Interaction with Calmodulin

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1074/JBC.M112.383299

Editorial overview --New frontiers of biophysical methods: Tools for structural biology and beyond

Publisher: Elsevier BV

Date: 10-2014

DOI: 10.1016/J.SBI.2014.09.005

Reliable structural interpretation of small-angle scattering data from bio-molecules in solution - The importance of quality control and a standard reporting framework

Publisher: Springer Science and Business Media LLC

Date: 17-05-2012

DOI: 10.1186/1472-6807-12-9

Abstract: Small-angle scattering is becoming an increasingly popular tool for the study of bio-molecular structures in solution. The large number of publications with 3D-structural models generated from small-angle solution scattering data has led to a growing consensus for the need to establish a standard reporting framework for their publication. The International Union of Crystallography recently established a set of guidelines for the necessary information required for the publication of such structural models. Here we describe the rationale for these guidelines and the importance of standardising the way in which small-angle scattering data from bio-molecules and associated structural interpretations are reported.

Activation of Myosin Light Chain Kinase Requires Translocation of Bound Calmodulin

Publisher: Elsevier BV

Date: 02-2001

DOI: 10.1074/JBC.C000857200

Differences in the heme environment of soybean leghemoglobin components shown by 1H-NMR spectroscopy

Publisher: Elsevier BV

Date: 1982

DOI: 10.1016/0167-4838(82)90094-2

Neutron Resonance Scattering Shows Specific Binding of Plutonium to the Calcium-Binding Sites of the Protein Calmodulin and Yields Precise Distance Information

Publisher: American Chemical Society (ACS)

Date: 06-1997

DOI: 10.1021/JA9633124

Corrigendum to “Calmodulin Disrupts the Structure of the HIV-1 MA Protein” [J. Mol. Biol. 400/4 (2010) 702–714]

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1016/J.JMB.2011.09.003

Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints

Publisher: Springer Science and Business Media LLC

Date: 16-11-2007

DOI: 10.1007/S10858-007-9211-5

Abstract: Determination of the accurate three-dimensional structure of large proteins by NMR remains challenging due to a loss in the density of experimental restraints resulting from the often prerequisite perdeuteration. Solution small-angle scattering, which carries long-range translational information, presents an opportunity to enhance the structural accuracy of derived models when used in combination with global orientational NMR restraints such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). We have quantified the improvements in accuracy that can be obtained using this strategy for the 82 kDa enzyme Malate Synthase G (MSG), currently the largest single chain protein solved by solution NMR. Joint refinement against NMR and scattering data leads to an improvement in structural accuracy as evidenced by a decrease from approximately 4.5 to approximately 3.3 A of the backbone rmsd between the derived model and the high-resolution X-ray structure, PDB code 1D8C. This improvement results primarily from medium-angle scattering data, which encode the overall molecular shape, rather than the lowest angle data that principally determine the radius of gyration and the maximum particle dimension. The effect of the higher angle data, which are dominated by internal density fluctuations, while beneficial, is also found to be relatively small. Our results demonstrate that joint NMR/SAXS refinement can yield significantly improved accuracy in solution structure determination and will be especially well suited for the study of systems with limited NMR restraints such as large proteins, oligonucleotides, or their complexes.

Neutron Diffraction And Scattering

Publisher: Wiley

Date: 15-01-2002

DOI: 10.1002/047120918X.EMB0999

Effects of gadolinium chelate on the evolution of the nanoscale structure in peptide hydrogels

Publisher: Wiley

Date: 23-06-2011

DOI: 10.1002/BIP.21689

The Structure of the KinA-Sda Complex Suggests an Allosteric Mechanism of Histidine Kinase Inhibition

Publisher: Elsevier BV

Date: 04-2007

DOI: 10.1016/J.JMB.2007.01.064

Abstract: The Bacillus subtilis histidine kinase KinA controls activation of the transcription factor governing sporulation, Spo0A. The decision to sporulate involves KinA phosphorylating itself on a conserved histidine residue, after which the phosphate moiety is relayed via two other proteins to Spo0A. The DNA-damage checkpoint inhibitor Sda halts this pathway by binding KinA and blocking the autokinase reaction. We have performed small-angle X-ray scattering and neutron contrast variation studies on the complex formed by KinA and Sda. The data show that two Sda molecules bind to the base of the DHp dimerization domain of the KinA dimer. In this position Sda does not appear to be able to sterically block the catalytic domain from accessing its target histidine, as previously proposed, but rather may effect an allosteric mode of inhibition involving transmission of the inhibitory signal via the four-helix bundle that forms the DHp domain.

Coassembling Peptide-Based Biomaterials:  Effects of Pairing Equal and Unequal Chain Length Oligopeptides

Publisher: American Chemical Society (ACS)

Date: 18-11-2006

DOI: 10.1021/CM061071L

Lev Feigin (1928–2022)

Publisher: International Union of Crystallography (IUCr)

Date: 24-06-2022

DOI: 10.1107/S205327332200626X

Corrigendum to A picornaviral loop-to-loop replication complex [J. Struct. Biol. 166 (2009) 251-262] (DOI:10.1016/j.jsb.2009.02.010)

Publisher: Elsevier BV

Date: 11-2009

DOI: 10.1016/J.JSB.2009.06.008

The Conformationally Dynamic C Helix of the RIα Subunit of Protein Kinase A Mediates Isoform-specific Domain Reorganization upon C Subunit Binding

Publisher: Elsevier BV

Date: 10-2005

DOI: 10.1074/JBC.M506769200

Structural themes and variations in protein kinase A as seen by small-angle scattering and neutron contrast variation

Publisher: Springer Science and Business Media LLC

Date: 20-04-2006

DOI: 10.1007/S00249-006-0061-Y

Abstract: Using small-angle solution scattering and neutron contrast variation, we have studied the structure of the multi-subunit protein kinase A. We have gained insights into how nature can take a set of common structural domains (or themes) and modulate their interactions via sequence variations and second messenger mediated signaling to affect enzyme activity and receptor binding important for targeting this multi-function enzyme to specific sub-cellular locations. These studies demonstrate the power of neutron contrast variation to expand our knowledge of the dynamic supra-molecular structures that carry out biological function.

The Macromolecular Architecture of Extracellular Domain of αNRXN1: Domain Organization, Flexibility, and Insights into Trans-Synaptic Disposition

Publisher: Elsevier BV

Date: 08-2010

DOI: 10.1016/J.STR.2010.06.005

Structures of Calmodulin and a Functional Myosin Light Chain Kinase in the Activated Complex:  A Neutron Scattering Study

Publisher: American Chemical Society (ACS)

Date: 05-1997

DOI: 10.1021/BI9702703

Biological Small-Angle Scattering: Theory and Practice. By Eaton E. Lattman, Thomas D. Grant and Edward H. Snell. Oxford University Press, 2018. Pp. 288. Price GBP 65.00, hardback, ISBN 9780199

Publisher: International Union of Crystallography (IUCr)

Date: 2019

DOI: 10.1107/S2059798318015917

Small-angle scattering and 3D structure interpretation

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1016/J.SBI.2016.05.003

Abstract: This review focuses on advances in the application of solution small-angle scattering (SAS) in structural analysis of biomolecules and the complexes they form. Ex les highlighted illustrate the unique contribution of SAS, using both X-rays and neutrons, in hybrid or integrative modelling methods. The increased information content when neutron scattering with contrast variation is used is a particular focus. Finally, progress toward an agreed reporting framework, the development of open data and model archives, and the importance of these initiatives is covered.

Activation of the Retroviral Budding Factor ALIX

Publisher: American Society for Microbiology

Date: 09-2011

DOI: 10.1128/JVI.02653-10

Abstract: The cellular ALIX protein functions within the ESCRT pathway to facilitate intralumenal endosomal vesicle formation, the abscission stage of cytokinesis, and enveloped virus budding. Here, we report that the C-terminal proline-rich region (PRR) of ALIX folds back against the upstream domains and auto-inhibits V domain binding to viral late domains. Mutations designed to destabilize the closed conformation of the V domain opened the V domain, increased ALIX membrane association, and enhanced virus budding. These observations support a model in which ALIX activation requires dissociation of the autoinhibitory PRR and opening of the V domain arms.

Ca²⁺‐induced structural changes in phosphorylase kinase detected by small‐angle X‐ray scattering

Publisher: Wiley

Date: 04-2005

DOI: 10.1110/PS.041124705

Structural properties of a haemophore facilitate targeted elimination of the pathogen Porphyromonas gingivalis

Publisher: Springer Science and Business Media LLC

Date: 05-10-2018

DOI: 10.1038/S41467-018-06470-0

Abstract: Porphyromonas gingivalis is a keystone bacterial pathogen of chronic periodontitis. P . gingivalis is unable to synthesise the porphyrin macrocycle and relies on exogenous porphyrin, including haem or haem biosynthesis intermediates from host sources. We show that under the iron-limited conditions prevailing in tissue environments, P . gingivalis expresses a haemophore-like protein, HusA, to mediate the uptake of essential porphyrin and support pathogen survival within epithelial cells. The structure of HusA, together with titration studies, mutagenesis and in silico docking, show that haem binds in a hydrophobic groove on the α-helical structure without the typical iron coordination seen in other haemophores. This mode of interaction allows HusA to bind to a variety of abiotic and metal-free porphyrins with higher affinities than to haem. We exploit this unusual porphyrin-binding activity of HusA to target a prototypic deuteroporphyrin-metronidazole conjugate with restricted antimicrobial specificity in a Trojan horse strategy that effectively kills intracellular P. gingivalis .

Crystal structure of TTHA0988 in space group P43212

Publisher: Worldwide Protein Data Bank

Date: 24-11-2010

DOI: 10.2210/PDB3OEP/PDB

C-terminal domain of KipI from Bacillus subtilis

Publisher: Worldwide Protein Data Bank

Date: 20-01-2009

DOI: 10.2210/PDB2ZP2/PDB

Troponin I Inhibitory Peptide (96−115) Has an Extended Conformation When Bound to Skeletal Muscle Troponin C

Publisher: American Chemical Society (ACS)

Date: 05-1999

DOI: 10.1021/BI990150Q

A Novel Structure of an Antikinase and its Inhibitor

Publisher: Elsevier BV

Date: 2011

DOI: 10.1016/J.JMB.2010.10.047

Abstract: In Bacillus subtilis, the KipI protein is a regulator of the phosphorelay governing the onset of sporulation. KipI binds the relevant sensor histidine kinase, KinA, and inhibits the autophosphorylation reaction. Gene homologues of kipI are found almost ubiquitously throughout the bacterial kingdom and are usually located adjacent to, and often fused with, kipA gene homologues. In B. subtilis, the KipA protein inhibits the antikinase activity of KipI thereby permitting sporulation. We have used a combination of biophysical techniques in order to understand the domain structure and shape of the KipI-KipA complex and probe the nature of the interaction. We also have solved the crystal structure of TTHA0988, a Thermus thermophilus protein of unknown function that is homologous to a KipI-KipA fusion. This structure, which is the first to be described for this class of proteins, provides unique insight into the nature of the KipI-KipA complex. The structure confirms that KipI and KipA are proteins with two domains, and the C-terminal domains belong to the cyclophilin family. These cyclophilin domains are positioned in the complex such that their conserved surfaces face each other to form a large "bicyclophilin" cleft. We discuss the sequence conservation and possible roles across species of this near-ubiquitous protein family, which is poorly understood in terms of function.

Ligand-induced Conformational Changes via Flexible Linkers in the Amino-terminal region of the Inositol 1,4,5-Trisphosphate Receptor

Publisher: Elsevier BV

Date: 11-2007

DOI: 10.1016/J.JMB.2007.08.057

Abstract: Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP(3)R have been reported those of the IP(3)-binding core (IP(3)R(CORE)) with bound IP(3), and the suppressor domain. This study examines the structural effects of ligand binding on an IP(3)R construct, designated IP(3)R(N), that contains both the IP(3)-binding core and the suppressor domain. Our circular dichroism results reveal that the IP(3)-bound and IP(3)-free states have similar secondary structure content, consistent with preservation of the overall fold within the in idual domains. Thermal denaturation data show that, while IP(3) has a large effect on the stability of IP(3)R(CORE), it has little effect on IP(3)R(N), indicating that the suppressor domain is critical to the stability of IP(3)R(N). The NMR data for IP(3)R(N) provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP(3)-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP(3)R(N) undergoes a change in average conformation in response to IP(3) binding and the presence of Ca2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP(3) binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set.

Histidine Kinase Regulation by a Cyclophilin-like Inhibitor

Publisher: Elsevier BV

Date: 12-2008

DOI: 10.1016/J.JMB.2008.09.017

Abstract: The sensor histidine kinase A (KinA) from Bacillus subtilis triggers a phosphorelay that activates sporulation. The antikinase KipI prevents sporulation by binding KinA and inhibiting the autophosphorylation reaction. Using neutron contrast variation, mutagenesis, and fluorescence data, we show that two KipI monomers bind via their C-domains at a conserved proline in the KinA dimerization and histidine-phosphotransfer (DHp) domain. Our crystal structure of the KipI C-domain reveals the binding motif has a distinctive hydrophobic groove formed by a five-stranded antiparallel beta-sheet a characteristic of the cyclophilin family of proteins that bind prolines and often act as cis-trans peptidyl-prolyl isomerases. We propose that the DHp domain of KinA transmits conformational signals to regulate kinase activity via this proline-mediated interaction. Given that both KinA and KipI homologues are widespread in the bacterial kingdom, this mechanism has broad significance in bacterial signal transduction.

Conformational Differences Among Solution Structures of the Type Iα, IIα and IIβ Protein Kinase A Regulatory Subunit Homodimers: Role of the Linker Regions

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1016/J.JMB.2004.02.028

Small-Angle Neutron Scattering with Contrast Variation Reveals Spatial Relationships between the Three Subunits in the Ternary Cardiac Troponin Complex and the Effects of Troponin I Phosphorylation

Publisher: American Chemical Society (ACS)

Date: 04-06-2003

DOI: 10.1021/BI0341509

Abstract: Small-angle neutron scattering with contrast variation has been used to determine the shapes and dispositions of the three subunits of cardiac troponin and to study the influence of phosphorylation on the structure. Three contrast variation series were collected on three different isotopically labeled variants of the cTnC/cTnI/cTnT(198-298) complex, one of which contained deuterated and bisphosphorylated cTnI. Analysis of the scattering data shows cTnT(198-298) interacting with a single lobe of a somewhat compacted cTnC that sits at one end of an elongated rodlike cTnI, covering about one-third of its length. The cTnT(198-298) sits near the center of the long cTnI axis. The components undergo significant conformational changes and reorientations in response to protein kinase A phosphorylation of cTnI. The rodlike cTnI bends sharply at the end interacting with the cTnC/cTnT(198-298) component, which reorients so as to maintain its contacts with cTnI while undergoing only a relatively small change in shape.

Ritva Serimaa (1957–2016)

Publisher: International Union of Crystallography (IUCr)

Date: 02-2017

DOI: 10.1107/S1600576716019609

Mutation in a flexible linker modulates binding affinity for modular complexes

Publisher: Wiley

Date: 09-03-2019

DOI: 10.1002/PROT.25675

Abstract: Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which β-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.

2023 update of template tables for reporting biomolecular structural modelling of small-angle scattering data

Publisher: International Union of Crystallography (IUCr)

Date: 02-2023

DOI: 10.1107/S2059798322012141

Abstract: In 2017, guidelines were published for reporting structural modelling of small-angle scattering (SAS) data from biomolecules in solution that exemplified best-practice documentation of experiments and analysis. Since then, there has been significant progress in SAS data and model archiving, and the IUCr journal editors announced that the IUCr biology journals will require the deposition of SAS data used in biomolecular structure solution into a public archive, as well as adherence to the 2017 reporting guidelines. In this context, the reporting template tables accompanying the 2017 publication guidelines have been reviewed with a focus on making them both easier to use and more general. With input from the SAS community via the IUCr Commission on SAS and attendees of the triennial 2022 SAS meeting (SAS2022, C inas, Brazil), an updated reporting template table has been developed that includes standard descriptions for proteins, glycosylated proteins, DNA and RNA, with some reorganization of the data to improve readability and interpretation. In addition, a specialized template has been developed for reporting SAS contrast-variation (SAS-cv) data and models that incorporates the additional reporting requirements from the 2017 guidelines for these more complicated experiments. To demonstrate their utility, ex les of reporting with these new templates are provided for a SAS study of a DNA–protein complex and a SAS-cv experiment on a protein complex. The ex les demonstrate how the tabulated information promotes transparent reporting that, in combination with the recommended figures and additional information best presented in the main text, enables the reader of the work to readily draw their own conclusions regarding the quality of the data and the validity of the models presented.

Neutron-Scattering Studies Reveal Further Details of the Ca²⁺/Calmodulin-Dependent Activation Mechanism of Myosin Light Chain Kinase^†

Publisher: American Chemical Society (ACS)

Date: 15-09-1998

DOI: 10.1021/BI981311D

Further Insights into Calmodulin−Myosin Light Chain Kinase Interaction from Solution Scattering and Shape Restoration

Publisher: American Chemical Society (ACS)

Date: 22-08-2003

DOI: 10.1021/BI0348664

Abstract: We have gained new insight into the interactions between the second-messenger protein calmodulin (CaM) and myosin light chain kinase from skeletal muscle (skMLCK) using small-angle solution scattering and shape restoration. Specifically, we explored the nature of a 2Ca(2+)-CaM-skMLCK complex and compared it to a 4Ca(2+)-CaM-skMLCK complex under the same conditions. The 2Ca(2+) complex has been proposed to be physiologically relevant. To aid in the interpretation of the data, we developed a shape restoration approach, implemented in GA_STRUCT, that combines many of the best features of other available methods into a single, automated package. Importantly, GA_STRUCT explicitly addresses the problem of the existence of multiple solutions to the inverse scattering problem and produces a consensus envelope from a set of shapes that fit the input intensity. Small-angle scattering intensity profiles measured or calculated from known structures were used to test GA_STRUCT, which was then used to generate low-resolution models for three complexes: 2Ca(2+)-CaM-skMLCK, 4Ca(2+)-CaM-skMLCK, and 4Ca(2+)-CaM-skMLCK with a bound substrate. These models were used in conjunction with high-resolution structures of the protein components to better understand the interactions among them. In the case of the 2Ca(2+)-CaM-skMLCK complex, the consensus envelope is consistent with CaM in a fully collapsed state with its two globular lobes in close contact with each other while the catalytic cleft of the kinase is open. The consensus envelope for the 4Ca(2+)-CaM-skMLCK complex indicates that the collapsed CaM has swung further away from the open catalytic cleft of the skMLCK than in the 2Ca(2+) complex, and further that substrate binding to this complex results in closure of the kinase catalytic cleft, in agreement with previous neutron scattering results. These results indicate that activation of MLCK by CaM can only occur once CaM is fully translocated away from the catalytic cleft, which is presumably linked to full release of the pseudo-substrate/inhibitory sequence. Our scattering data indicate that this step is completed only when all four calcium binding sites are loaded.

A Model Structure of the Muscle Protein Complex 4Ca2+.cntdot.Troponin C.cntdot.Troponin I Derived from Small-Angle Scattering Data: Implications for Regulation

Publisher: American Chemical Society (ACS)

Date: 1994

DOI: 10.1021/BI00209A011

Abstract: We report here a model structure for 4Ca2+.troponin C.troponin I derived from small-angle X-ray and neutron scattering data using a Monte Carlo modeling method. In this model, troponin I appears as a spiral structure that wraps around 4Ca2+.troponin C which adopts an extended dumbbell conformation similar to that observed in the crystal structures of troponin C. The troponin I spiral has the approximate dimensions of an alpha-helix and winds through the hydrophobic "cups" in each globular domain of troponin C. The model is consistent with a body of previously published biochemical data on the interactions between troponin C and troponin I, and suggests the molecular mechanism for the Ca(2+)-sensitive switch that regulates the muscle contraction/relaxation cycle involves a signal transmitted via the central spiral region of troponin I.

Reflections of a Woman Scientist in the Year 2011

Publisher: CSIRO Publishing

Date: 2011

DOI: 10.1071/CH11035

Evolution of Quaternary Structure in a Homotetrameric Enzyme

Publisher: Elsevier BV

Date: 07-2008

DOI: 10.1016/J.JMB.2008.05.038

Abstract: Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits.

The C0C1 Fragment of Human Cardiac Myosin Binding Protein C Has Common Binding Determinants for Both Actin and Myosin

Publisher: Elsevier BV

Date: 11-2011

DOI: 10.1016/J.JMB.2011.09.026

Abstract: The N-terminal domains of cardiac myosin binding protein C (MyBP-C) play a regulatory role in modulating interactions between myosin and actin during heart muscle contraction. Using NMR spectroscopy and small-angle neutron scattering, we have determined specific details of the interaction between the two-module human C0C1 cMyBP-C fragment and F-actin. The small-angle neutron scattering data show that C0C1 spontaneously polymerizes monomeric actin (G-actin) to form regular assemblies composed of filamentous actin (F-actin) cores decorated by C0C1, similar to what was reported in our earlier four-module mouse cMyBP-C actin study. In addition, NMR titration analyses show large intensity changes for a subset of C0C1 peaks upon addition of G-actin, indicating that human C0C1 interacts specifically with actin and promotes its assembly into filaments. During the NMR titration, peaks corresponding to cardiac-specific C0 domain are the first to be affected, followed by those from the C1 domain. No peak intensity or position changes were detected for peaks arising from the disordered proline/alanine-rich (P/A) linker connecting C0 with C1, despite previous suggestions of its involvement in binding actin. Of considerable interest is the observation that the actin-interaction "hot-spots" within the C0 and C1 domains, revealed in our NMR study, overlap with regions previously identified as binding to the regulatory light chain of myosin and to myosin ΔS2. Our results suggest that C0 and C1 interact with myosin and actin using a common set of binding determinants and therefore support a cMyBP-C switching mechanism between myosin and actin.

Insights into biomolecular function from small-angle scattering

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1016/S0959-440X(97)80081-4

Abstract: Recent advances in neutron and X-ray sources and instrumentation, new and improved scattering techniques, and molecular biology techniques, which have permitted facile preparation of s les, have each led to new opportunities in using small-angle scattering to study the conformations and interactions of biological macromolecules in solution as a function of their properties. For ex le, new instrumentation on synchrotron sources has facilitated time-resolved studies that yield insights into protein folding. More powerful neutron sources, combined with molecular biology tools that isotopically label s les, have facilitated studies of biomolecular interactions, including those involving active enzymes.

Neutron Diffraction Studies of Bacteriorhodopsin Structure

Publisher: Springer US

Date: 1984

DOI: 10.1007/978-1-4899-0375-4_14

Global Conformational Changes Control the Reactivity of Methane Monooxygenase

Publisher: American Chemical Society (ACS)

Date: 05-1999

DOI: 10.1021/BI982991N

Solution Structures of Calcium Regulating Proteins: A Small-Angle Scattering Study

Publisher: Informa UK Limited

Date: 03-1990

DOI: 10.1080/00268949008025787

Publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution

Publisher: International Union of Crystallography (IUCr)

Date: 17-05-2012

DOI: 10.1107/S0907444912012073

1H N.M.R Studies of High-Spin Complexes of Soybean Leghemoglobin. Interactions Between the Distal Histidine and Acetate, Formate and Fluoride Ligands

Publisher: CSIRO Publishing

Date: 1986

DOI: 10.1071/CH9860317

Abstract: High-resolution 1H n.m.r . spectra have been measured for the acetate, formate and fluoride complexes of ferric soybean leghaemoglobin a. The chemical shifts of the resolved ow-field haem resonances are very sensitive to pH, reflecting titration of a group, assigned as the distal histidine, with pKa 7.1, 7.3 and 6.2 for the acetate, formate and fluoride complexes respectively. The magnitude of the pH-induced resonance shifts indicates direct interaction between the distal histidine and the axial ligand . The hyperfine shifted resonances of ferrous deoxyleghaemoglobin, in which no sixth ligand is expected, are insensitive to pH over the range pH 5.5-9.3. Evidence for conformational mobility of the distal histidine is presented.

Domain organization of the monomeric form of the Tom70 mitochondrial import receptor

Publisher: Elsevier BV

Date: 05-2009

DOI: 10.1016/J.JMB.2009.03.070

Abstract: Tom70 is a mitochondrial protein import receptor composed of 11 tetratricopeptide repeats (TPRs). The first three TPRs form an N-terminal domain that recruits heat shock protein family chaperones, while the eight C-terminal TPRs form a domain that receives, from the bound chaperone, mitochondrial precursor proteins destined for import. Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) analysis characterized Tom70 as an elongated monomer. A model for the Tom70 monomer was proposed based on the alternate interpretation of the domain pairings observed in the crystal structure of the Tom70 dimer and refined against the SAXS data. In this "open" model of the Tom70 monomer, the chaperone- and precursor-binding sites are exposed and lay side by side on one face of the molecule. Fluorescence anisotropy measurements indicated that monomeric Tom70 can bind both chaperone and precursor peptides and that chaperone peptide binding does not alter the affinity of Tom70 for the precursor peptide. SAXS was unable to detect any shape change in Tom70 upon chaperone binding. However, molecular modeling indicated that chaperone binding is incompatible with Tom70 dimer formation. It is proposed that the Tom70 monomer is the functional unit mediating initial chaperone docking and precursor recognition.

The different views from small angles

Publisher: Proceedings of the National Academy of Sciences

Date: 04-2008

DOI: 10.1073/PNAS.0801324105

A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

Publisher: Wiley

Date: 12-03-2009

DOI: 10.1002/BIT.22300

Abstract: The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 15 degrees C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with K(i) values of modified savinases sixfold higher than the unmodified savinase. Modeling of small-angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold-active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme-formulation.

Protein kinase A targeting and activation as seen by small-angle solution scattering

Publisher: Elsevier BV

Date: 07-2006

DOI: 10.1016/J.EJCB.2006.01.003

Abstract: We have studied the solution structures of the multi-functional protein kinase A using small-angle X-ray and neutron scattering and have found a remarkable structural ersity in the different isoforms of this multi-subunit enzyme, in spite of its having high sequence homology and a common domain organization within its sequences. The available high-resolution crystal and NMR structural data for the protein kinase A components have aided in the interpretation of the solution scattering data and enabled us to develop models that bring insights into protein kinase A activation and targeting mechanisms, such as the opening and closing of the catalytic cleft to facilitate substrate binding or inhibition, respectively, and the role of sequence segments that join functional domains in the R subunit in providing a structurally flexible scaffold for interactions with the C subunit and A kinase-anchoring proteins (AKAPs).

The Role of Heme Binding by DNA-protective Protein from Starved Cells (Dps) in the Tolerance of Porphyromonas gingivalis to Heme Toxicity

Publisher: Elsevier BV

Date: 12-2012

DOI: 10.1074/JBC.M112.392787

Small‐Angle Scattering of Neutrons and X‐Rays

Publisher: Wiley

Date: 16-02-2015

DOI: 10.1002/9780470015902.A0003047.PUB3

Calmodulin Disrupts the Structure of the HIV-1 MA Protein

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.JMB.2010.05.022

C Subunits Binding to the Protein Kinase A RIα Dimer Induce a Large Conformational Change

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1074/JBC.M313405200

Small‐angle Scattering of Neutrons and X‐rays

Publisher: Wiley

Date: 15-01-2010

DOI: 10.1002/9780470015902.A0003047.PUB2

Changes in Small-Angle X-ray Scattering Parameters Observed upon Binding of Ligand to Rabbit Muscle Pyruvate Kinase Are Not Correlated with Allosteric Transitions

Publisher: American Chemical Society (ACS)

Date: 27-07-2010

DOI: 10.1021/BI100147W

Synaptic Arrangement of the Neuroligin/β-Neurexin Complex Revealed by X-Ray and Neutron Scattering

Publisher: Elsevier BV

Date: 06-2007

DOI: 10.1016/J.STR.2007.04.010

Inelastic neutron scattering in biology

Publisher: Elsevier BV

Date: 1986

DOI: 10.1016/S0378-4363(86)80129-2

Refinement of Multidomain Protein Structures by Combination of Solution Small-Angle X-ray Scattering and NMR Data

Publisher: American Chemical Society (ACS)

Date: 03-11-2005

DOI: 10.1021/JA054342M

Abstract: Determination of the 3D structures of multidomain proteins by solution NMR methods presents a number of unique challenges related to their larger molecular size and the usual scarcity of constraints at the interdomain interface, often resulting in a decrease in structural accuracy. In this respect, experimental information from small-angle scattering of X-ray radiation in solution (SAXS) presents a suitable complement to the NMR data, as it provides an independent constraint on the overall molecular shape. A computational procedure is described that allows incorporation of such SAXS data into the mainstream high-resolution macromolecular structure refinement. The method is illustrated for a two-domain 177-amino-acid protein, gammaS crystallin, using an experimental SAXS data set fitted at resolutions from approximately 200 A to approximately 30 A. Inclusion of these data during structure refinement decreases the backbone coordinate root-mean-square difference between the derived model and the high-resolution crystal structure of a 54% homologous gammaB crystallin from 1.96 +/- 0.07 A to 1.31 +/- 0.04 A. Combining SAXS data with NMR restraints can be accomplished at a moderate computational expense and is expected to become useful for multidomain proteins, multimeric assemblies, and tight macromolecular complexes.

Small-Angle Scattering and Neutron Contrast Variation for Studying Bio-Molecular Complexes

Publisher: Humana Press

Date: 2009

DOI: 10.1007/978-1-59745-483-4_20

Abstract: Structural molecular biology over the past several decades has progressed from studies of the in idual proteins, subunits, and domains that accomplish specific biochemistry to seeking to understand the dynamic bio-molecular complexes and assemblies that are responsible for biological function. This progress has led to an expansion of the structural analysis "tool box" to include methods that complement the mainstay techniques of the field: X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy. Small-angle scattering of X-rays or neutrons is one such complementary technique that provides information on the size and shape of scattering particles in solution. This low-resolution structural information can be a powerful complement to high-resolution structural data, especially for the study of bio-molecular interactions with ligands or each other. Further, exploitation of the different neutron-scattering properties of the stable isotopes of hydrogen ((1)H and (2)H) can be used to enrich the information available from the small-angle scattering data, especially for bio-molecular complexes.

1H, 13C and 15N backbone and side chain resonance assignments of the N-terminal domain of the histidine kinase inhibitor KipI from Bacillus subtilis

Publisher: Springer Science and Business Media LLC

Date: 04-06-2010

DOI: 10.1007/S12104-010-9237-6

Abstract: KipI is a sporulation inhibitor in Bacillus subtilis which acts by binding to the dimerisation and histidine phosphotransfer (DHp) domain of KinA, the principle input kinase in the phosphorelay responsible for sporulation. The (15)N, (13)C and (1)H chemical shift assignments of the N-terminal domain of KipI were determined using multidimensional, multinuclear NMR experiments. The N-terminal domain has two conformers and resonance assignments have been made for both conformers.

Linköpings Universitet

Location: Sweden

View Organisation

Los Alamos National Laboratory

Location: United States of America

View Organisation

Australian Nuclear Science And Technology Organisation

Location: Australia

View Organisation

University Of New South Wales

Location: Australia

View Organisation

Los Alamos National Laboratory Bioscience Division

Location: United States of America

View Organisation

University Of Sydney

Location: Australia

View Organisation

Yale University

Location: United States of America

View Organisation

University Of Utah

Location: United States of America

View Organisation

(Shared Resource For Protein Discovery)

Start Date: 2011

End Date: 2011

Funder: Australian Research Council

(Australian Synchrotron Access Program)

Start Date: 2012

End Date: 2016

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0770321

Start Date: 01-2007

End Date: 01-2010

Amount: $390,000.00

Funder: Australian Research Council

Federation Fellowships - Grant ID: FF0457488

Start Date: 07-2005

End Date: 06-2010

Amount: $1,519,710.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100148

Start Date: 2017

End Date: 12-2018

Amount: $300,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120103841

Start Date: 06-2012

End Date: 06-2015

Amount: $315,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100171

Start Date: 06-2011

End Date: 12-2011

Amount: $200,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0668496

Start Date: 05-2006

End Date: 05-2007

Amount: $950,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0883032

Start Date: 02-2009

End Date: 12-2010

Amount: $1,300,000.00

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

Discovery Projects - Grant ID: DP0984536

Start Date: 06-2009

End Date: 06-2012

Amount: $420,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR120200004

Start Date: 07-2012

End Date: 12-2015

Amount: $30,000,000.00

Funder: Australian Research Council