Principal Supervisor: Dr. Allister Crow, Department of Pathology, University of Cambridge
Co-supervisor: Professor David Roper, School of Life Sciences
PhD project title: Structural and functional investigation of bacterial cytochrome c maturation
University of Registration: University of Warwick
The c-type cytochromes are an important class of metalloproteins that contain a covalently-bound heme molecule attached to two cysteine residues located within a classical amino acid sequence motif, CXXCH. C-type cytochromes are key components of many membrane-integral respiratory complexes and perform essential electron transfer roles in bacterial respiratory chains.
In Gram-positive bacteria, cyanobacteria, and plant chloroplasts (but not humans), just three dedicated proteins (ResA, ResB and ResC) are required for cytochrome assembly. ResA is membrane-anchored thiodisulfide oxidoreductase responsible for maintaining the cysteine residues of apo-cytochromes in a reduced state. ResB and ResC are intrinsic membrane proteins dedicated to the export of heme across the cytoplasmic membrane and its subsequent covalent attachment to the CXXCH motif.
This project seeks to understand how c-type cytochromes are assembled in Gram-positive bacteria. The project will use a mixture of X-ray crystallography, microbiology and biophysical methods and will address key biological questions on how cytochrome maturation works. For example: How is heme transferred across the bacterial membrane? How is covalent attachment of heme catalysed? What do ResB and ResC look like and how might they have evolved? Do cytochrome maturation proteins operate independently, or do they cluster together as a large ‘cytochrome maturase’ complex?
Understanding cytochrome c maturation will not only reveal important details of a fundamental biological process underpinning numerous important bacterial respiratory chains, but may also reveal how these proteins could be inhibited. Bacterial cytochrome c maturation systems are very different from those of humans and animals so drugs targeted toward the Res proteins could find use as novel antibiotics.
The project will build upon previous work on the structure and biophysical properties of ResA (Crow 2004, Crow 2005, Lewin 2006) and BdbD (Crow 2009).
Protocols are already in place for producing all components of the cytochrome c maturation system and both ResB and ResC have been purified to homogeneity allowing work to commence immediately. We will seek crystal structures of the Res proteins and in parallel use simple microbiological experiments to investigate how they work. Interaction between the Res proteins will be assessed using co-purification methods and isothermal titration calorimetry and key residues thought to be functionally important will be identified using site-directed mutagenesis coupled with established in vivo techniques for detection of cytochromes. It may also be possible to reconstitute cytochrome c maturation in vitro.
Outcomes: The project will provide structural and mechanistic insight into how c-type cytochromes are made in Gram-positive bacteria. Knowledge of the structural underpinnings of cytochrome maturation may present new opportunities for design of novel antimicrobial compounds. The project will imbue students with valuable skills in protein purification, structure determination, and biophysical characterisation.
Structural basis of redox-coupled protein substrate selection by the cytochrome c biosynthesis protein ResA
A Crow, RM Acheson, NE Le Brun, A Oubrie
Journal of Biological Chemistry (2004)
The role of ResA in type II cytochrome c maturation
A Crow, NE Le Brun, A Oubrie
Biochemical Society Transactions 33 (1), 149-151 (2005)
Molecular basis for specificity of the extracytoplasmic thioredoxin ResA
A Lewin, A Crow, A Oubrie, NE Le Brun
Journal of Biological Chemistry 281 (46), 35467-35477 (2006)
Crystal structure and biophysical properties of Bacillus subtilis BdbD An oxidizing thiol: disulfide oxidoreductase containing a novel metal site
A Crow, A Lewin, O Hecht, MC Möller, GR Moore, L Hederstedt, NE Le Brun
Journal of Biological Chemistry 284 (35), 23719-23733 (2009)
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
Purification of both soluble and membrane proteins.
Use of X-ray crystallography to determine macromolecular structures.
Development of structure-based hypotheses of protein mechanism and testing of these ideas using in vitro and in vivo techniques.
Additional methods may include simple DNA work such as cloning genes for expression in bacteria and site-directed mutagenesis to dissect structure-function relationships. Spectroscopy and gel-based techniques will be used for detection of assembled cytochromes.
Contact: Dr. Allister Crow, Department of Pathology, University of Cambridge