Cambridge team uncover membrane protein tool

The team, led by Leopold Ilag of the University of Cambridge, have developed a mass spectrometry-based approach to analysing membrane p roteins and identifying drug binding to them.

Membrane proteins are prime drug targets, but are known to be extremely difficult to work with because their structures are stabilised by both hydrophilic and hydrophobic molecular forces.

While scientists understand much about the structure and stability of water-soluble proteins that 'float' around in humans, they know very little about the shape and stability of proteins that are embedded, or folded, into the lipid membrane of cells. Such membrane proteins make up about 30 per cent of all proteins in the body.

Indeed, the majority of drugs on the market today are effective because they work on membrane proteins, but the basic knowledge about these proteins lags far behind that of water-soluble proteins. This means that designing new drugs to target them has mostly been like playing a game of blind man's buff, with hits comoing more by luck than judgement.

This situation has prompted the formation of a scientific collaboration - the Membrane Protein Structure Initiative - to improve the understanding of the structure and function of membrane proteins.

In their study, the Cambridge researchers examined a complex formed between the membrane protein EmrE - a transporter that transfers drugs and other compounds across bacterial cell membranes - and the lipid dodecylmaltoside (DDM) using mass spectrometry. This analytical method would ordinarily be discounted for this type of analysis, because of the unfavourable, hydrophobic environment within the mass spectrometer.

Despite this, the scientists showed that it was possible to preserve the structure of the protein in its lipid carrier (micelle), even when it was converted to the gas phase for injection into the mass spectrometer.

Usually, the peaks observed by these complexes are very broad and heterogeneous, reflecting the varied distribution of lipid molecules attached to the protein complex. As such, the spectrum cannot be interpreted.

It is important that membrane proteins remain in their usual state in order to preserve functional integrity, including the binding of small-molecule ligands that could act as drugs. This has meant that researchers have had to cope with the confounding issue of sample heterogeneity, in the form of native and exogenous lipids and detergents.

Ilag and colleagues used a compound that binds to EmrE - called tetraphenyl phosphonium (TPP+) - to mimic a drug/membrane protein interaction, such as might be used in a drug screening assay.

"To simplify this complexity we used a tandem mass spectrometry procedure [that revealed] clusters of DDM molecules, as well as sequential release of TPP(+) and EmrE from the complex as the collision cell voltage is raised. Taken together, the results provide direct evidence for drug binding within a relevant gas-phase protein-micelle complex,"​ said the researchers.

Tandem mass spectrometry led to the identification of a broad peak that contained protein, detergent, and ligand, with these components being dissociable from the parent complex in reverse order as the collision voltage in the mass spectrometer was increased.

The team is using the approach to examine the functions of membrane proteins involved in mutlidrug resistance. They describe the work in the Journal of the American Chemical Society (10 November issue).

Other teams have explored ways to make membrane proteins more amenable to research. For example, earier this year a US team from the University of Virginia Health System came up with a protocol to extract proteins from membranes by using chemicals that allow them to be reversibly folded and refolded. The proteins can then be studied using crystallography or nuclear magnetic resonance imaging.

The Virginia researchers reported their findngs in the Proceedings of the National academy of Sciences (23 Merch 2004).