Protein NMR

Dr C J Craven

The research in my group is mainly in the application of solution state NMR (and also structural bioinformatic techniques and X-ray crystallography) to study the structure, dynamics and interactions of proteins and ligands.

Recent work includes:
[1] Development of the combinatorial selective labelling method [1] for obtaining very rapid assignments of 15N HSQC spectra. The method is based on using different combinations of selective labels incorporated using a cell free translation system. The method offers the potential of opening up the use of chemical shift mapping in HSQC spectra to many more researchers, since the problem of resonance assignment is rendered semi-trivial.

[2] Backbone NMR assignment of the 394 residue (43 kDa) protein PGK using a very efficient semi automated assignment method [2]. This is one of the largest single chain proteins assigned to date. The results enabled us to analyse the effect of domain dissection on the folding and stability properties of this protein.

[3] Delineation of a novel family of human proteins, the PLUNC proteins [3], which are candidate host defence proteins. Expression studies show that these proteins are largely confined to the upper respiratory tract. Sequence analysis indicates a strong relationship to the BPI family of proteins, which are implicated in the response to bacterial LPS. This relationship is much clearer when the proteins are analysed at the secondary structure level, rather than by simple sequence comparison. Comparison of the equivalent human and mouse proteins (orthologues) indicates that the proteins are evolving rapidly, which is a frequent chracteristic of proteins involved in host defence.

[4] Demonstration, using NMR, of 3D domain swapping in the human cystatin A dimer [4].

[5] Determination, by NMR, of the first structure of any member of the TCTP proteins [5]. This is a family of proteins that is conserved across the eukaryotic kingdom, and is very highly abundant, yet for which no clear function has been proven. Our structure suggests a link at the structural level to the protein MSS4, which is an accessory protein for small GTPases.

Figure: Ray-traced representation of the 2D 15N HSQC spectrum of TCTP. The intense signals towards the centre of the spectrum arise from a well conserved mobile loop of twenty residues.

Selected Publications

Dimerization of protein G B1 domain at low pH: A conformational switch caused by loss of a single hydrogen bond. Tomlinson JH, Craven CJ, Williamson MP, Pandya MJ. Proteins. 2009 Dec 21. In press.

Structural tightening and interdomain communication in the catalytic cycle of phosphoglycerate kinase. Marston JP, Cliff MJ, Reed MA, Blackburn GM, Hounslow AM, Craven CJ, Waltho JP. J Mol Biol. 2010 Feb 19;396(2):345-60.

Dimerisation of the UBA domain of p62 inhibits ubiquitin binding and regulates NF-kappaB signalling. Long J, Garner TP, Pandya MJ, Craven CJ, Chen P, Shaw B, Williamson MP, Layfield R, Searle MS. J Mol Biol. 2010 Feb 12;396(1):178-94.

Automated protein structure calculation from NMR data. Williamson MP, Craven CJ. J Biomol NMR. 2009 Mar;43(3):131-43.

Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad. Nyon MP, Rice DW, Berrisford JM, Hounslow AM, Moir AJ, Huang H, Nathan S, Mahadi NM, Bakar FD, Craven CJ. J Mol Biol. 2009 Jan 9;385(1):226-35.

The denatured state of N-PGK is compact and predominantly disordered. Cliff MJ, Craven CJ, Marston JP, Hounslow AM, Clarke AR, Waltho JP. J Mol Biol. 2009 Jan 9;385(1):266-77.

Crystallization and preliminary X-ray analysis of recombinant Glomerella cingulata cutinase. Nyon MP, Rice DW, Berrisford JM, Huang H, Moir AJ, Craven CJ, Nathan S, Mahadi NM, Abu Bakar FD. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jun 1;64(Pt 6):504-8

Exclusion of the native alpha-helix from the amyloid fibrils of a mixed alpha/beta protein. Morgan GJ, Giannini S, Hounslow AM, Craven CJ, Zerovnik E, Turk V, Waltho JP, Staniforth RA. J Mol Biol. 2008 Jan 11;375(2):487-98.

Structural and functional analysis of RNA and TAP binding to SF2/ASF. Tintaru AM, Hautbergue GM, Hounslow AM, Hung ML, Lian LY, Craven CJ, Wilson SA. EMBO Rep. 2007 Aug;8(8):756-62.

Assignment of 1H, 13C, and 15N resonances for SF2 RNA recognition motif 2. Tintaru AM, Hautbergue GM, Hounslow AM, Lian LY, Wilson SA, Craven CJ. J Biomol NMR. 2007 Jun;38(2):193.

A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK. Cliff MJ, Alizadeh T, Jelinska C, Craven CJ, Staniforth RA, Waltho JP. J Mol Biol. 2006 Dec 8;364(4):810-23.

Multiple forms of copper (II) co-ordination occur throughout the disordered N-terminal region of the prion protein at pH 7.4. Wells MA, Jelinska C, Hosszu LL, Craven CJ, Clarke AR, Collinge J, Waltho JP, Jackson GS. Biochem J. 2006 Dec 15;400(3):501-10.

A reassessment of copper(II) binding in the full-length prion protein. Wells MA, Jackson GS, Jones S, Hosszu LL, Craven CJ, Clarke AR, Collinge J, Waltho JP. Biochem J. 2006 Nov 1;399(3):435-44.

Feo--transport of ferrous iron into bacteria. Cartron ML, Maddocks S, Gillingham P, Craven CJ, Andrews SC. Biometals. 2006 Apr;19(2):143-57.

The denatured state under native conditions: a non-native-like collapsed state of N-PGK. Reed MA, Jelinska C, Syson K, Cliff MJ, Splevins A, Alizadeh T, Hounslow AM, Staniforth RA, Clarke AR, Craven CJ, Waltho JP. J Mol Biol. 2006 Mar 24;357(2):365-72.

A Combinatorial Selective Labeling Method for the Assignment of Backbone Amide NMR Resonances. Parker, M. J.,Aulton-Jones, M., Hounslow, A. M., Craven, C. J. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY; 2004; 126(16); 5020-5021.

Effects of domain dissection on the folding and stability of the 43 kDa protein PGK probed by NMR. Reed, M. A. C., Hounslow, A. M., Sze, K. H., Barsukov, I. G., Hozzu, L., L., P., Clarke, A. R., Craven, C. J., and Waltho, J. P. JOURNAL OF MOLECULAR BIOLOGY, 2003, 330 1189-1201.

PLUNC: A novel family of candidate host defence proteins expressed in the upper airways and nasopharynx. Bingle, C. D., and Craven, C. J. HUMAN MOLECULAR GENETICS, 2002, 11 937-943

Three-dimensional domain swapping in the folded and molten- globule states of cystatins, an amyloid-forming structural superfamily. Staniforth R. A., Giannini S., Higgins L. D., Conroy M. J., Hounslow A. M., Jerala R., Craven C. J. and Waltho J. P. EMBO JOURNAL, 2001, 20 4774-4781.

Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones. Thaw P., Baxter N. J., Hounslow A. M., Price C., Waltho J. P. and Craven C. J. NATURE STRUCTURAL BIOLOGY, 2001, 8 701-704.