Mapping lipid and detergent binding sites on the surface of membrane proteins

When membrane proteins are crystallised and X-ray crystallography is used to determine their 3D structure, non protein molecules can often be seen surrounding their hydrophobic regions. These molecules could either be lipids or detergent molecules, that were used to isolate the proteins from their membranes. Unfortunately though, it is usually the case that only parts of these molecules are 'seen', due to disorder, and so it is not possible to decide which ones are which. We are going to use a novel method which will allow us to unambiguously resolve this identification issue. We will synthesise heavy atom tagged lipids and detergents and add these to the crystals. The extra X-ray scattering power of these heavy atoms will allow us to clearly distinguish which is which. The results of this study will be very useful in helping to understand the molecular details of how membrane proteins interact with lipids. In many cases these interactions are actually essential for the protein to function correctly. In X-ray crystallography in order to solve 3D structures the diffraction data must be combined with the 'phase' information. These heavy atoms 'tagged' lipids and detergents will be tested to see if they can provide a new general method for 'phasing' diffraction data with crystals of membrane proteins.

There is a great deal of current interest in trying to understand the molecular details of how membrane proteins interact with lipids. It is frustrating therefore that many crystal structures of membrane proteins so reveal non-proton electron densities around the protein's hydrophobic surfaces but usually only part of the lipids or detergents are seen, and so these electron densities cannot be unambiguous assigned. In this proposal we will use a novel approach to solve the problem by co-crystallising with heavy-duty atom tagged lipids and detergents. The extra scattering power of the heavy atom will allow us to definitively assign these electron densities to either lipids or detergents, and to study the details of their binding sites. Use of these heavy atoms tagged lipids and detergents could in principle allow the 'phases' to be solved and to provide a new general route to 'phasing' crystal structures of membrane proteins. We will fully explore this possibility.