Oxford University Department of Biochemistry
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Anthony Watts
Resolving details of membrane peptides and proteins at high resolution

Co-workers: Lubica Aslimovska, Helen Attrill, Olivia Berthoumieu, Peter Fisher, Peter Harding, Peter Judge, Julia Koeppe, Liz Mitchell, Simon Ross, Satita Tapaneeyakorn, Mohamed Triba, Krisztina Varga

Resolving structural details of membrane receptors is still a major challenge, not least because of the difficulties of expression, crystallization and their size. Since membrane protein secondary structure can be modelled for many cases, we have been devising solid state NMR methods for determining high resolution (sub-Å) details of information-rich sites within membrane receptors. In particular we have resolved new information about ligand (drug, neurotransmitter or solute) binding sites, and related all this to functional description. Our recent focus is on the neurotensin receptor (NTS1) which we have expressed in E. coli in structural biology amounts in a functionally competent form for structural studies, some of which involve single molecule approaches for bionanotechnological and drug design applications.

Having developed novel solid state NMR approaches to give detailed information about ligands at their site of action in wild type membrane targets (Watts, 2005), we have now extended our interest to GPCRs.


Genomically, membrane proteins constitute a significant part (30-40%) of higher life form genomes, but proteomically they are only around 1-2%, making purification and characterization difficult.

We are now focussing on a major class of receptors, those with putatively 7 transmembrane domains as predicted from computational methods. One example, the neurotensin receptor, NTS1, is now available highly purified monodispersed in detergent and in a ligand-binding form. One approach to monitoring ligand binding has been to develop a novel surface plasmon resonance method for tagging the natural ligand, neurotensin (13-mer peptide) to the chip and monitoring protein binding (Harding et al., 2006). Fluorescently (FPs) tagged NTS1 is now being used in fluorescence resonance energy transfer methods to resolve long range information of protein-protein signalling. Solid state NMR and EM are used to investigate ligand structure and protein structure.


Research Images

Substituted imidazo-pyridines are inhibitors of the gastric H+/K+-ATPase. By specific labelling of members of this drug family with NMR visible isotopes, we have been able to define the full conformation of the bound ligand, and suggest mechanism for inhibition from homology modelling with a related protein. (Kim, Watts & Watts, 2005, J. Med. Chem. 48, 7145-7152 and Watts, Watts & Middleton, 2001, J. Biol. Chem., 276, 43197-43204).

The cation- p interaction of acetyl choline, a major brain neurotransmitter, and the ligand gated, nicotinic acetyl choline receptor has been resolved using solid state NMR, giving an insight into the binding mechanism and the residues surrounding the site. (Watts, 2005, Nature Reviews Drug Discovery , 4, 555-568

The way in which retinal is restrained within its binding site in membrane-embedded mammalian rhodopsin, and the structural details of the site, have been resolved for the early activation states of this light-activated GPCR, using high resolution solid state NMR to measure atomic distances within the retinal to high accuracy (+/- 0.2Å). (Spooner, et al., 2004, J. Mol. Biol., 343, 719-730)

Contact: anthony.watts@bioch.ox.ac.uk

Graduate Student and Postdoctoral Positions: Enquiries with CV welcome

Website: http://www4.bioch.ox.ac.uk/~oubsu/group/awatts.htm

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