Folding and stability of the beta adrenergic receptor

Proteins are the 'worker molecules' in life. Cells in our bodies sense and communicate with the outside world via proteins embedded in membranes that surround the cells. This project aims to design a new method to study a particular type of protein, membrane proteins. In particular, it is focussed on a protein that acts as a receptor during communication between cells. This receptor is a member of the largest family of membrane proteins in the human genome, a family that is the major target for the development of new medicines. Unfortunately, there is a limited understanding of how these membrane receptor proteins work at a molecular level. This is because they are notoriously unstable outside the membrane and it is difficult to obtain sufficient amounts for scientific study. Recent advances are, however, beginning to alter this situation. We aim to devise new methods to study a phenomenon known as 'folding'. Genes carry the code for proteins, but puzzles remain in deciphering how genetic information is translated into functional proteins. Proteins begin as a string of amino acids, which then have to fold-up to a particular shape in the right part of the body. If this folding fails, disaster strikes and the proteins malfunction. The most glaring gaps in knowledge of this folding phenomenon come with membrane proteins. We propose to forge new territory by devising new methods to understand membrane protein folding. We intend to work out what makes the receptor unstable and investigate how to ensure it folds correctly.

G protein coupled receptors are the largest family of membrane proteins in the human genome and dominate targets for the development of new drugs. A major challenge in biomolecular research of receptors is to fold and stabilise the proteins. We propose to study the denaturation process and devise methods to re-fold and stabilise the beta adrenergic receptor. Research into G protein coupled receptors is at an exciting stage, with the recent determination of high resolution structures of a human beta adrenergic receptor. Moreover, we and our collaborators have stabilised another beta adrenergic receptor through mutagenesis and solved the structure. This thermostable mutant receptor has tremendous potential in a folding study as the increased stability and reduced conformational heterogeneity greatly improve the chances of successful refolding. Refolding becomes an even better prospect in the lipid bicelle system we will use. We have shown that bicelles dramatically improve the stability of a G protein coupled receptor and they are a highly successful refolding system for helical membrane proteins in general. Moreover, bicelles were used to obtain crystals of the beta receptor. A tactic we will employ with the bicelles is to alter the properties off their lipid bilayer-disc to favour activated conformations of the beta adrenergic receptor. We will use our experience in devising lipid folding systems to create bicelle environments that stabilise and fold different conformations of the receptor. Different mutants will also allow us to probe the role of particular structural regions during denaturation, unfolding and refolding. We believe GPCR folding is a tractable problem. The employment of bicelles and mutants that stabilise the receptor, together with knowledge of the denaturation mechanism, affords a very good opportunity to identify key receptor structural losses and prevent or reverse them.