Award details

Structure determination of the 7-helix transmembrane protein receptor pSRII by solution NMR

Reference	BB/G011915/1
Principal Investigator / Supervisor	Professor Daniel Nietlispach
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Biochemistry
Funding type	Research
Value (£)	376,499
Status	Completed
Type	Research Grant
Start date	01/04/2009
End date	31/03/2012
Duration	36 months

Abstract

A multitude of problems make structural studies of membrane proteins a substantial challenge. Membrane proteins play key functional roles and their dysfunction is directly involved in many diseases. Elucidating the structure of membrane proteins is invaluable for understanding their function. Given the extensive difficulties, encouraging advances have been made recently using X-ray crystallography and NMR in solution and solid. Solution-state NMR spectroscopy is rapidly winning importance in the study of structure and dynamics of integral membrane proteins but so far has primarily concentrated on the more accessible b-barrel porin proteins with only few smaller a-helical proteins studied. We will study a helical transmembrane protein as a representative of a wider group of key membrane protein receptors. Our immediate goal is to determine the structure of the seven-helix transmembrane protein pSRII solubilized in a detergent micelle. We will concentrate on the development of the NMR methodology required to study such large protein-detergent systems (50-70 kDa). The feasibility of our approach is backed by our recent breakthrough results achieved over the last 12 months where we show the complete sequential assignment of this 7TM receptor. In addition, we will map the interaction of pSRII with its downstream transducer HtrII. Our techniques will be generally applicable to the study of other helical membrane proteins in solution. We will use the full potential of NMR and study protein dynamics and modes of detergent interaction and assess how these are governed by detergent choice. How far the properties of functional membrane proteins in their natural lipid environment can be correctly reproduced in detergent micelles has been the focus of a longstanding debate. Based on structure and dynamics we will be able to compare our findings with the results on pSRII available from X-ray and solid-state NMR. All our objectives will be met within the grant period of 36 months.

Summary

Most living organisms are assembled from a very large number of cells. Each of these cells is a specialist that contributes through a large number of activities to the support of an organism. Cells are composed mostly of a liquid interior that accommodates the precious machinery including the building blocks of life and all the information to produce proteins. A water insoluble protective lipid layer called a membrane surrounds the cell. To function properly and also when necessary to adjust the interior workings of the cell to changing external demands the cell needs to be continuously supplemented with nutrients and instructions that come from the outside. To help with this, a large number of molecules, called membrane proteins such as e.g. ligand receptors are embedded in the lipid layer, where they span the membrane. They are the liaison centres of the cell and communicate between interior and exterior worlds to shuttle information and material across the cell-interface. Their involvement in so many complex information transfer processes and their implication in a large number of diseases have made them prime targets for the majority of existing drugs on the market. Biologists and chemists study the properties of such molecules in order to better understand the cellular and biomolecular processes. One important aspect is to understand the three-dimensional organization of these molecules. While this can be obtained quite routinely and with great success for water-soluble molecules, it is much more difficult to achieve for membrane embedded proteins using the traditional structure determination techniques of X-ray crystallography and nuclear magnetic resonance (NMR). Consequently structure determination of insoluble membrane proteins is still in its early stages. Our proposal aims at the development of the NMR technique to solve the structure of such membrane proteins. We concentrate our studies on a particular member, for us a model system that belongs to afamily of proteins with many analogies to receptors. The latter have proven particularly evasive to structural studies so far and our research efforts will improve this situation.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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