ORCID Profile
0000-0003-2972-5235
Current Organisation
University of York
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Publisher: American Chemical Society (ACS)
Date: 21-03-2006
DOI: 10.1021/BI052605J
Abstract: Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly, the K42E mutation produces a number of changes at the heme center (nearly 20 A distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies, suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c' in vivo.
Publisher: Springer Science and Business Media LLC
Date: 10-02-2015
DOI: 10.1007/S10482-015-0400-Z
Abstract: The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.
Publisher: Cold Spring Harbor Laboratory
Date: 24-12-2020
DOI: 10.1101/2020.12.24.424174
Abstract: Changes at the cell surface enable bacteria to survive in dynamic environments, such as erse niches of the human host. Here, we reveal “Periscope Proteins” as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure thus variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly-available long-read genome sequencing data, we propose this new class could contain over 50 distinct proteins, including those implicated in host colonisation and biofilm formation by human pathogens. In large multi-domain proteins, sequence ergence between adjacent domains appears to reduce inter-domain misfolding. Periscope Proteins break this “rule”, suggesting their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria and the immune system.
Publisher: Portland Press Ltd.
Date: 02-2005
DOI: 10.1042/BST0330187
Abstract: Cytochrome c′, a c-type cytochrome with unique spectroscopic and magnetic properties, has been characterized in a variety of denitrifying and photosynthetic bacteria. Cytochrome c′ has a role in defence and/or removal of NO but the mechanism of action is not clear. To examine the function of cytochrome c′ from Neisseria meningitidis, the protein was purified after heterologous overexpression in Escherichia coli. The electronic spectra of the oxidized c′ demonstrated a pH-dependent transition (over the pH range of 6–10) typical of known c′-type cytochromes. Interestingly, the form in which NO is supplied determines the redox state of the resultant haem-nitrosyl complex. Fe(III)–NO complexes were formed when Fe(II) or Fe(III) cytochrome c′ was sparged with NO gas, whereas an Fe(II)–NO complex was generated when NO was supplied using DEA NONOate (diazeniumdiolate).
Publisher: Elsevier
Date: 2008
Publisher: American Chemical Society (ACS)
Date: 26-05-2005
DOI: 10.1021/BI050428G
Abstract: The heme coordination chemistry and spectroscopic properties of Rhodobacter capsulatus cytochrome c' (RCCP) have been compared to data from Alcaligenes xylosoxidans (AXCP), with the aim of understanding the basis for their different reactivities with nitric oxide (NO). Whereas ferrous AXCP reacts with NO to form a predominantly five-coordinate heme-nitrosyl complex via a six-coordinate intermediate, RCCP forms an equilibrium mixture of six-coordinate and five-coordinate heme-nitrosyl species in approximately equal proportions. Ferrous RCCP and AXCP both exhibit high Fe-His stretching frequencies (227 and 231 cm(-)(1), respectively), suggesting that factors other than the Fe-His bond strength account for their differences in heme-nitrosyl coordination number. Resonance Raman spectra of ferrous-nitrosyl RCCP confirm the presence of both five-coordinate and six-coordinate heme-NO complexes. The six-coordinate heme-nitrosyl of RCCP exhibits a fairly typical Fe-NO stretching frequency (569 cm(-)(1)), in contrast to the relatively high value (579 cm(-)(1)) of the AXCP six-coordinate heme-nitrosyl intermediate. It is proposed that NO experiences greater steric hindrance in binding to the distal face of AXCP, as compared to RCCP, leading to a more distorted Fe-N-O geometry and an elevated Fe-NO stretching frequency. Evidence that RCCP has a more accessible distal coordination site than in AXCP stems from the fact that ferric RCCP readily forms a heme complex with exogenous imidazole, whereas AXCP does not. A model is proposed in which distal heme-face accessibility, rather than the proximal Fe-His bond strength, determines the heme-nitrosyl coordination number in cytochromes c'.
Publisher: Proceedings of the National Academy of Sciences
Date: 31-05-2021
Abstract: The structure of single and tandem SHIRT domains from the streptococcal surface protein Sgo_0707 were determined. In conjunction with biophysics and molecular dynamics simulations, the results show that the observed gene length variation would result in differential projection of the host ligand binding domain on the bacterial cell surface. An analysis of long-read DNA sequence data reveals many other repetitive bacterial surface proteins that appear to undergo gene length variation. We propose that these variable-length “Periscope Proteins” represent an important mechanism of bacterial cell surface modification with potential roles in infection and immune evasion.
Publisher: Elsevier BV
Date: 2007
DOI: 10.1016/J.PEP.2006.06.026
Abstract: The purification of small quantities of a major small c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis has recently been reported. In order to characterise this protein further we have expressed the gene encoding this cytochrome in Escherichia coli and have purified the protein to homogeneity. The protein is directed to the E. coli periplasm using its native signal sequence suggesting that it may be translocated via a Sec-type system in K. stuttgartiensis. The cytochrome has the visible spectroscopic properties typical of a low-spin c-type cytochrome, but these spectroscopic features broaden in high salt solutions. The oxidised cytochrome was able to bind the ligands NO and cyanide. A redox potential of +230 mV suggests that the protein is suitable to act as an electron carrier protein that may be involved in the respiratory chain between hydrazine oxidation and the reduction of nitrite. The predicted protein sequence for the cytochrome suggests it to be a predominantly alpha-helical protein, and this is supported by circular dichroism.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for James Moir.