Structural Biology

Dr J B Rafferty

My research interests centre on the structural study of proteins and DNA primarily by X-ray crystallography and electron microscopy. The work provides detailed 3-dimensional insights into biological macromolecules and their assemblies that can be combined with biochemical and genetic investigations to provide a better understanding of how they function.

Figure Legend : Model of binding of RusA resolvase to a DNA Holliday junction based on the data from the protein:DNA complex described in reference MacMaster et al. Nucleic Acids Res (2006). Location of active site residues on protein indicated by pink spheres and sites of cleavage on DNA indicated by orange spheres.

Currently my work is focussed on the elucidation of the molecular details underlying DNA recombination, in particular the protein components involved in processing DNA Holliday junctions, on a structural understanding of the helicases and accessory proteins involved in the initiation of DNA replication and on the structures of DNA transcriptional regulators. In addition, I have a longstanding interest in the enzymes of fatty acid biosynthesis. My group is also embarking upon research into multi-protein complexes from eukaryotes.

The first area saw the successful structure determination of the E.coli DNA recombination protein RuvA and its complex bound to its DNA target that consists of a fourway crossover of DNA duplexes known as a Holliday junction. Recently, we have focussed on the resolution of the junction catalysed by various resolvase enzymes and determined the structures of the bacterial enzymes RusA and RecU that cut the DNA at the Holliday junction crossover point in a sequence specific manner. We also study gene transcription factors from bacteria and plants and performed the first structural characterization of proteins from the Lrp / AsnC regulator family. This has now been extended to structure determinations of two more family members and investigation of how particular family members might have roles beyond transcriptional control, including accessory functions in DNA recombination.

Eukaryotic proteins form elaborate complex networks to regulate cellular development via processes such as gene silencing and are a major and challenging structural target. We are developing our structural interests in this area alongside studies of large multi-protein complexes extracted from yeast cells using a novel tagging protocol that does not involve recombinant protein overexpression and should hopefully reveal information on the native interacting protein networks in vivo.

Selected Publications

Studies of Toxoplasma gondii and Plasmodium falciparum enoyl acyl carrier protein reductase and implications for the development of antiparasitic agents. Muench SP, Prigge ST, McLeod R, Rafferty JB, Kirisits MJ, Roberts CW, Mui EJ, Rice DW. Acta Crystallogr D Biol Crystallogr. 2007 Mar;63(Pt 3):328-38.

Structural studies of fatty acyl-(acyl carrier protein) thioesters reveal a hydrophobic binding cavity that can expand to fit longer substrates. Roujeinikova A, Simon WJ, Gilroy J, Rice DW, Rafferty JB, Slabas AR. J Mol Biol. 2007 Jan 5;365(1):135-45.

RusA Holliday junction resolvase: DNA complex structure--insights into selectivity and specificity. Macmaster R, Sedelnikova S, Baker PJ, Bolt EL, Lloyd RG, Rafferty JB. Nucleic Acids Res. 2006;34(19):5577-84.

Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family. Thaw P, Sedelnikova SE, Muranova T, Wiese S, Ayora S, Alonso JC, Brinkman AB, Akerboom J, van der Oost J, Rafferty JB. Nucleic Acids Res. 2006 Mar 9;34(5):1439-49.

Leonard PM, Smits SHJ, Sedelnikova SE, Brinkman AB, de Vos WM, van der Oost J, Rice, DW, Rafferty JB. Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus. EMBO J. (2001) 20 990-997.

Bailey S, Sedelnikova SE, Mesa P, Ayora S, Waltho JP, Ashcroft AE, Baron AJ, Alonso JC, Rafferty JB. Structural analysis of Bacillus subtilis SPP1 phage helicase loader protein G39P. J. Biol. Chem. (2003) 278, 15304-15312.

McGregor N, Ayora S, Sedelnikova S, Carrasco B, Alonso JC, Thaw P, Rafferty, J. The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage. Structure (2005) 13, 1341-1351.

Thaw P, Sedelnikova SE, Muranova T, Wiese S, Ayora S, Alonso JC, Brinkman AB, Akerboom J, van der Oost J, Rafferty JB. Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family. Nucleic Acids Res. (2006) 34, 1439-1449.

MacMaster R, Sedelnikova SE, Bolt EL, Lloyd RG, Baker PJ, Rafferty JB. The structure of a RusA Holliday junction resolvase: DNA complex provides insights into structural selectivity and sequence specificity during the resolution step of homologous recombination. Nucleic Acids Res. (2006)