Using nuclear magnetic resonance spectroscopy in structural biology
(1) Protein structure
A major theme is the determination of protein structures in solution using NMR spectroscopy. Recent work has highlighted crucial inter-molecular contacts between modules in the molecular assembly line of the polyketide synthase that makes 6-deoxyerythronolide B, a precursor of the antibiotic erythromycin A. Future PhD projects include further studies of PKS and NRPS multizenzyme complexes and several protein components of an important signal transduction cascade in T-lymphocytes.
(2) Backbone dynamics
The relaxation properties of backbone amide 15N nuclei are sensitive to overall protein motions on the nanosecond time scale and to local motions on time scales ranging from picoseconds to milliseconds. For example, the 15N transverse relaxation rates of atrial natriuretic peptide (ANP), a small unstructured hormone, depend mostly on interactions with neighbouring residues and can be predicted by a simple model of segmental motion. The formation of a disulphide bridge between Cys7 and Cys23 has a dramatic effect on the relaxation rates of adjacent sites. The changes in conformational entropy between the oxidised and reduced states of ANP account for the different affinities of these peptides for the dimeric receptor NPRA. Future PhD projects will include further studies of the properties of natively unfolded proteins.
The chemical shifts of protein 1H, 13C and 15N nuclei are exquisitely sensitive to local changes in electronic environments that occur when different backbone and side-chain conformations are adopted. We have been developing new methods for deconvoluting these complex relationships, using the amino acid sequence of a target protein and experimental shifts of 13Ca, 13Cb, 13C', 1Ha and 15N nuclei to make predictions of the backbone dihedral angles phi and psi. This work has yielded a new software package (called DASH) that is able to convert the experimental chemical shifts of backbone nuclei into dihedral angle restraints for protein structure calculations. Future PhD projects will extend this approach.
(4) Membrane proteins
We are developing several new approaches for studying membrane proteins using NMR and electrospray mass spectrometry. Future PhD projects in this area include applications to proteins from the Influenza A virus, cytolytic delta-endotoxins and ligand gated ion channels.
(1) Peto et al. (2004) Backbone dynamics of oxidised and reduced forms of human atrial natriuretic peptide. J. Struct. Biol. 146 (in press).
(2) Kolocouris et al. (2004) Interaction between an amantadine analog and the transmembrane portion of the Influenza A M2 protein in liposomes probed by 1H NMR spectroscopy of the ligand. J. Med. Chem. 47 (in press).
(3) Broadhurst et al. (2003) The structure of docking domains in modular polyketide synthases. Chem. Biol. 10, 731.
(4) Hansen et al (2002) Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 13, 1376.