Dr John Rafferty
Reader in Structural Biology
Principal Admissions Tutor
Tel: 0114 222 2809
My research interests centre on structural studies of proteins and nucleic acids primarily by X-ray crystallography but also utilizing other biophysical techniques such as NMR, SAXS 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. Currently my research includes determining structural details of proteins involved in DNA or RNA binding; proteins from bacterial membrane transport systems; toxin & antitoxin proteins; and glycosidic enzymes.
DNA & RNA metabolism
Drawing originally from work on E.coli DNA recombination protein RuvA and its DNA junction complex, my group has focussed on the recognition and processing of junction points in DNA. The work has involved studies of proteins from a diverse range of bacterial and viral sources. It has also expanded into structural studies of RNA binding proteins from bacterial pathogens and simple eukaryotes that regulate the assembly of the ribosome. In parallel with the crystallography, we have examined the solution structures by SAXS.
Recently we have carried out successful and exciting studies of proteins from the pathogen Campylobacter jejuni, which is the major cause of food poisoning worldwide. This work has seen structures determined for proteins from the bacterial periplasm responsible for metabolite transport across the inner membrane, protein folding & establishment of the outer membrane and mechanisms for avoiding the host immune system. The work has been extended to examine proteins from the periplasm of other organisms such as Rhodopseudomonas palustris, which is of interest because of its potential biotechnological role in lignin processing.
Glycoside recognition and processing
The carbohydrate coat on many cells provides both a protective role but also a nutrient source for invading organisms. We have been studying the structural determinants that control the way key enzymes and associated binding domains recognize specific targets and process them. These proteins in pathogenic organisms provide potentially excellent targets for inhibition and the development of antibacterial compounds.
Structural biology, X-ray crystallography, macromolecules, structure-function relationships
Level 3 Modules
MBB344 Genomic Science
Level 2 Modules
MBB261 Biochemistry 2
Level 1 Modules
MBB161 Biochemistry (Module Coordinator)
- Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila. Nature Communications, 10(1). View this article in WRRO
- The molecular basis of endolytic activity of a multidomain alginate lyase from Defluviitalea phaphyphila, a representative of a new lyase family, PL39. Journal of Biological Chemistry, 294(48), 18077-18091.
- A New Mechanism for High-Affinity Uptake of C4-Dicarboxylates in Bacteria Revealed by the Structure of Rhodopseudomonas palustris MatC (RPA3494), a Periplasmic Binding Protein of the Tripartite Tricarboxylate Transporter (TTT) Family. Journal of Molecular Biology, 431(2), 351-367.
- Structural basis for high-affinity adipate binding to AdpC (RPA4515), an orphan periplasmic-binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris. FEBS Journal. View this article in WRRO
- Structural insights into dynamics of RecU–HJ complex formation elucidates key role of NTR and stalk region toward formation of reactive state. Nucleic Acids Research, 45(2), 975-986. View this article in WRRO
- Direct observation of DNA threading in flap endonuclease complexes. Nature Structural and Molecular Biology, 23, 640-646. View this article in WRRO
- Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species.. Biochem J, 458(3), 499-511. View this article in WRRO
- Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry.. Mol Microbiol, 89(6), 1240-1258. View this article in WRRO
- Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase.. Nucleic Acids Res, 41(9), 5115-5126. View this article in WRRO
- The CouPSTU and TarPQM transporters in Rhodopseudomonas palustris: redundant, promiscuous uptake systems for lignin-derived aromatic substrates.. PLoS One, 8(3), e59844. View this article in WRRO
- Crystallization and preliminary X-ray analysis of the receiver domain of a putative response regulator, BPSL0128, from Burkholderia pseudomallei.. Acta Crystallogr Sect F Struct Biol Cryst Commun, 68(Pt 8), 917-922.
- The Stalk Region of the RecU Resolvase Is Essential for Holliday Junction Recognition and Distortion. J MOL BIOL, 410(1), 39-49.
- The virulence factor PEB4 (Cj0596) and the periplasmic protein Cj1289 are two structurally related SurA-like chaperones in the human pathogen Campylobacter jejuni.. J Biol Chem, 286(24), 21254-21265.
- Studies of Toxoplasma gondii and Plasmodium falciparum enoyl acyl carrier protein reductase and implications for the development of antiparasitic agents.. Acta Crystallogr D Biol Crystallogr, 63(Pt 3), 328-338. View this article in WRRO
- Structural studies of fatty acyl-(acyl carrier protein) thioesters reveal a hydrophobic binding cavity that can expand to fit longer substrates.. J Mol Biol, 365(1), 135-145.
- RusA Holliday junction resolvase: DNA complex structure--insights into selectivity and specificity.. Nucleic Acids Res, 34(19), 5577-5584. View this article in WRRO
- Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family.. Nucleic Acids Res, 34(5), 1439-1449. View this article in WRRO
- The Structure of Bacillus subtilis RecU Holliday Junction Resolvase and Its Role in Substrate Selection and Sequence-Specific Cleavage. Structure, 13(9), 1341-1351.
- Rapid analysis of protein-nucleic acid complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography.. J Biochem Biophys Methods, 58(1), 39-48.
- The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold.. Structure, 11(12), 1557-1567.
- Crystallization of RusA Holliday junction resolvase from Escherichia coli.. Acta Crystallogr D Biol Crystallogr, 59(Pt 12), 2262-2264.
- Non-cytotoxic variants of the Kid protein that retain their auto-regulatory activity. Plasmid, 50(2), 120-130.
- Structural basis of Synercid (quinupristin-dalfopristin) resistance in Gram-positive bacterial pathogens.. J Biol Chem, 278(32), 29963-29970.
- Expression, purification and crystallization of the Plasmodium falciparum enoyl reductase.. Acta Crystallogr D Biol Crystallogr, 59(Pt 7), 1246-1248.
- Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P.. Acta Crystallogr D Biol Crystallogr, 59(Pt 6), 1090-1092.
- Structural analysis of Bacillus subtilis SPP1 phage helicase loader protein G39P.. J Biol Chem, 278(17), 15304-15312.
- Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by site-directed mutagenesis. J BIOL CHEM, 278(7), 4435-4439.
- Squash glycerol-3-phosphate (1)-acyltransferase - Alteration of substrate selectivity and identification of arginine and lysine residues important in catalytic activity. J BIOL CHEM, 277(46), 43918-43923.
- The RuvABC resolvasome.. Eur J Biochem, 269(22), 5492-5501.
- A novel ligand-binding domain involved in regulation of amino acid metabolism in prokaryotes. J BIOL CHEM, 277(40), 37464-37468.
- Structural and functional analysis of the kid toxin protein from E. coli plasmid R1.. Structure, 10(10), 1425-1433.
- X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site.. Structure, 10(6), 825-835.
- The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms.. Structure, 10(4), 589-600.
- Crystallization of a complex of Caenorhabditis elegans diadenosine tetraphosphate hydrolase and a non-hydrolysable substrate analogue, AppCH2ppA.. Acta Crystallogr D Biol Crystallogr, 58(Pt 3), 526-528.
- Crystallization and preliminary X-ray crystallographic studies on acyl-(acyl carrier protein) from Escherichia coli.. Acta Crystallogr D Biol Crystallogr, 58(Pt 2), 330-332.
- Crystallization and preliminary X-ray crystallographic studies on the parD-encoded protein Kid from Escherichia coli plasmid R1.. Acta Crystallogr D Biol Crystallogr, 58(Pt 2), 355-358.
- Cloning, characterisation and crystallisation of a diadenosine 5 ',5 '''-P-1,P-4-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans. BBA-PROTEIN STRUCT M, 1550(1), 27-36.
- Functional studies of the Xenopus Polycomb-group proteins Xeed and XEz.. DEV BIOL, 235(1), 262-263.
- Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus.. Acta Crystallogr D Biol Crystallogr, 57(Pt 6), 886-888.
- A study of the structure-activity relationship for diazaborine inhibition of Escherichia coli enoyl-ACP reductase.. J Mol Biol, 309(1), 171-180.
- Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.. Structure, 9(5), 347-353.
- Crystallization and preliminary X-ray analysis of the glycerol-3-phosphate 1-acyltransferase from squash (Cucurbita moschata).. Acta Crystallogr D Biol Crystallogr, 57(Pt 3), 451-453.
- Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus.. EMBO J, 20(5), 990-997.
- Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I. INT J PARASITOL, 31(2), 109-113.
- Plant glycerol-3-phosphate-l-acyltransferase (GPAT): structure selectivity studies. Biochemical Society Transactions, 28(6), 677-679.
- Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity. Biochemical Society Transactions, 28(6), 680-681.
- The X-ray structure of Brassica napus beta-keto acyl carrier protein reductase and its implications for substrate binding and catalysis.. Structure, 8(4), 339-347.
- Crystallization of the NADP-dependent beta-keto acyl-carrier protein reductase from Brassica napus.. Acta Crystallogr D Biol Crystallogr, 56(Pt 1), 86-88.
- Crystallographic analysis of triclosan bound to enoyl reductase.. J Mol Biol, 294(2), 527-535.
- Inhibitor binding studies on enoyl reductase reveal conformational changes related to substrate recognition.. J Biol Chem, 274(43), 30811-30817.
- Molecular basis of triclosan activity. NATURE, 398(6726), 383-384.
- Molecular genetic analysis of enoyl-acyl carrier protein reductase inhibition by diazaborine. MOL MICROBIOL, 31(2), 443-450.
- Crystallization of Escherichia coli RuvA complexed with a synthetic Holliday junction.. Acta Crystallogr D Biol Crystallogr, 55(Pt 1), 263-265.
- The X-ray structure of Escherichia coli enoyl reductase with bound NAD+ at 2.1 A resolution.. J Mol Biol, 284(5), 1529-1546.
- Crystal structure of E.coli RuvA with bound DNA Holliday junction at 6 A resolution.. Nat Struct Biol, 5(6), 441-446.
- Mechanism of action of diazaborines.. Biochem Pharmacol, 55(10), 1541-1549.
- Crystallization of the NADP-dependent beta-keto acyl carrier protein reductase from Escherichia coli.. Acta Crystallogr D Biol Crystallogr, 54(Pt 3), 427-429.
- Structural similarities between Escherichia coli RuvA protein and other DNA-binding proteins and a mutational analysis of its binding to the holliday junction.. J Mol Biol, 278(1), 105-116.
- Insights into the mechanisms of homologous recombination from the structure of RuvA.. Curr Opin Struct Biol, 7(6), 798-803.
- Crystallization of E. coli RuvA gives insights into the symmetry of a Holliday junction/protein complex.. Acta Crystallogr D Biol Crystallogr, 53(Pt 1), 122-124.
- A mechanism of drug action revealed by structural studies of enoyl reductase.. Science, 274(5295), 2107-2110.
- Soluble and membrane bound components of plant lipid synthesis. CR ACAD SCI III-VIE, 319(11), 1043-1047.
- Crystallization of Escherichia coli enoyl reductase and its complex with diazaborine.. Acta Crystallogr D Biol Crystallogr, 52(Pt 6), 1181-1184.
- Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction.. Science, 274(5286), 415-421.
- Common themes in redox chemistry emerge from the X-ray structure of oilseed rape (Brassica napus) enoyl acyl carrier protein reductase.. Structure, 3(9), 927-938.
- The Met Repressor-Operator Complex: DNA Recognition by ?-Strands. Annals of the New York Academy of Sciences, 726(1 DNA Damage), 105-117.
- Crystallization of the NADH-specific enoyl acyl carrier protein reductase from Brassica napus.. J Mol Biol, 237(2), 240-242.
- Crystallization and initial X-ray analysis of the C2-subunit of crustacyanin. Journal of Molecular Biology, 224(1), 283-284.
- Cooperative tandem binding of met repressor of Escherichia coli. Nature, 341(6244), 711-715.
- Three-dimensional crystal structures of Escherichia coli met repressor with and without corepressor. Nature, 341(6244), 705-710.
- Crystallization of the met repressor from Escherichia coli. Journal of Molecular Biology, 200(1), 217-219.
- Fatty Acid Biosynthesis in Plants — Metabolic Pathways, Structure and Organization, Lipids in Photosynthesis (pp. 11-34). Springer Netherlands
- Determination of the X-Ray Crystallographic Structure of E.coli Butyryl-ACP, Advanced Research on Plant Lipids (pp. 139-142). Springer Netherlands
- Structural Studies on Plant and Bacterial Reductases Involved in Lipid Biosynthesis, Physiology, Biochemistry and Molecular Biology of Plant Lipids (pp. 38-41). Springer Netherlands
- Towards a Structural Understanding of Enzymes of Lipid Biosynthesis, Plant Lipid Metabolism (pp. 55-57). Springer Netherlands
Conference proceedings papers
- Structures of fatty acyl-( acyl carrier protein) thioesters reveal a binding cavity that expands to fit longer substrates. FEBS JOURNAL, Vol. 274 (pp 223-223)
- Crystal structure and mutational analysis of the diadenosine tetraphosphate (Ap4A) 'Nudix' hydrolase from Caenorhabditis elegans. DRUG DEVELOPMENT RESEARCH, Vol. 56(4) (pp 567-567)
- Acyltransferases and their role in the biosynthesis of lipids - opportunities for new oils. Journal of Plant Physiology, Vol. 158(4) (pp 505-513)
- CRYSTALLOGRAPHIC STUDIES OF THE NADP-DEPENDENT beta-KETO ACP REDUCTASE FROM ESCHERICHIA COLI. ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES, Vol. 55 (pp 293-294)