BA (Hons) Biochemistry, University of Oxford, 1991
D. Phil. Biochemistry, University of Oxford, 1994
The main component of research in this group is in the determination of macromolecular structures by X-ray crystallography. However, since not all proteins crystallize, every effort is made to complete our understanding of how proteins function by utilizing other methods, such as microbial genetics, monitoring protein:ligand interactions by biochemical and biophysical methods, electron microscopy and bioinformatics.
Variations in environmental factors, such as reduced aeration, extremes of temperature and the availability of essential nutrients, restrict microbial growth. As a result, bacteria spend much of their life cycle in stationary phase. The requirement for acclimatisation to the environment has forced bacteria to develop a series of complex adaptive responses to stress. One of the initial responses to stress of some Gram+ bacteria, including Bacillus subtilis, is to synthesise a large and diverse family of ?B-dependent general stress proteins. ?B is an alternative RNA polymerase subunit conferring promoter specificity thus directing gene expression in a defined manner.
?B is kept under strict control by the gene products found in the sigB operon, which may be functionally divided into "upstream" and "downstream" modules. Each module comprises an ATP-dependent serine/threonine protein kinase (RsbW, RsbT), a phospho-serine/phospho-threonine protein phosphatase (RsbU, RsbX), and a "switch" protein substrate for the kinase and phosphatase (RsbV, RsbS). In both modules, the kinases also participate in alternative protein:protein recognition events. Together, the binding partner for the kinase and the phosphorylation state of the switch molecules control the activity of ?B.
We utilize a multi-disciplinary approach to studying the proteins and their interactions with small and large ligands, with the focus on determining the X-ray crystal structures of the proteins and larger assemblies. For instance, with collaborators in the Microbiology Unit, University of Oxford, we have established the KM for ATP and KI for ADP of RsbW, the stoichiometry of the RsbW:?B and RsbW:RsbV complexes, the affinity of RsbW for its two alternative partners and the concentration of these three proteins in the cell, before and during stress. We have crystallized fragments of rsbU that encode the N-terminal or C-terminal domains, with crystals of N-RsbU diffracting X-rays to at least 1.6 Å. This structure has recently been solved by selenomethionyl MAD, revealing a dimeric structure comprised of two 4-helical bundles. We are replacing the functional copy of rsbU in the chromosome with a truncated form, encoding solely the catalytic domain, and monitoring ?B activity in the cell by fusing a reporter gene, lacZ, to a ?B-dependent gene. It is our belief that the function of N-RsbU is in molecular recognition and recruitment of the stimulatory factor, the kinase RsbT, and in the (de)regulation of the phosphatase activity of RsbU. We are determining the kinetic and equilibrium constants for RsbT and RsbU in order to map the RsbT-binding surface in RsbU. Intriguingly, the human pathogen Staphylococcus aureus encodes a ?B regulon that is similar to that of B. subtilis, but does not encode an RsbT orthologue, and thus the regulation of RsbU must differ in this organism. This is an interesting difference in physiology between phylogenetically closely-related members of the same bacterial group, which we will investigate biochemically as well as structurally.
In contrast, the alternative binding partner for RsbT is a large, approximately megadalton-sized complex formed between RsbR and RsbS, evidence for the existence of which in vivo is accumulating. We are examining this structure by cryo electron microscopy and single particle analysis. The differences between the structures of the RsbR:RsbS complex and the kinase-recruitment domain of RsbU in the context of molecular recognition by RsbT are the focus of current thinking. They contrast dramatically in terms of size. The likely functions of the RsbR paralogs in B. subtilis are the subject of further thought, and perhaps here bioinformatics will be a useful tool to study these proteins.
The structural biology group in the School of Cell and Molecular Biosciences is being established in the summer of 2003. We will have state-of-the art X-ray and computing facilities in a newly refurbished lab. The group will be small initially, and tightly focused, with expertise present in many aspects of structural biology. From October, it is expected to number 8 in total, comprising a mixture of PIs, postdocs, postgrads and a research technician. More details will be posted here when appropriate, including details of on-going collaborative research with colleagues within the school. There are no posts available at the moment, but informal enquiries to R.J.L. (firstname.lastname@example.org) are always welcome.
Wellcome Trust, BBSRC, Royal Society