Novel Detergent Systems for Membrane Protein Studies

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Detergents are essential tools for the biophysical study of membrane proteins, lipids, and other hydrophobic molecules. While traditional detergents remain in wide use and are essential reagents, no single detergent has ideal properties for all applications. As a result, several new alternative solubilization systems are opening up new avenues of research. The proposed program proposes to expand on three novel technologies that are being actively developed in the Privé lab. 1) Picodiscs. We have discovered that the small human protein Saposin A is able to form stable, defined, low-molecular weight discoidal complexes with various lipids. We have called these assemblies “picodiscs” in order to emphasize the similarities with the widely used “nanodiscs”. Nanodiscs have generated excitement as alternatives to detergents or bicelles for the stabilization of membrane proteins, however, nanodiscs suffer from a relatively high degree of polydispersity due to their size. Picodiscs, in contrast, are significantly smaller and thus have more defined stoicheometries. As a result, they can only accommodate smaller amphiphilic molecules such as specific lipids or single transmembrane alpha helices. Thus, picodiscs have unique properties and complement what is currently feasible with nanodiscs, and we will develop picodiscs as a unique solubilization system. 2) Lipopeptide detergents (LPDs). We have designed 20-25 amino acid polypeptides covalently coupled to fatty acyl chains as a new class of solubilizing amphiphile. LPDs can self-assemble into relatively rigid micelles with low aggregation numbers and can stabilize membrane proteins by accommodating these in the interior of cylindrical LPD micelles. We have designed and synthesized several series of LPDs, each with specific characteristics. LPDs readily crystallize, and we have determined high-resolution crystal structures of LPDs micelles containing 8, 9 and 12 chains arranged into cylindrical micelles with lipidic cores. We propose to refine and diversify the design of lipopeptides with novel properties, and to expand the potential of these novel amphiphiles into new applications. 3) SDS/MPD. We have found that some small alcohols, such as 2,4-methyl-pentanediol (MPD), have profound effects on the properties of ionic detergents. For example, SDS is normally a strong protein denaturant. However, mixtures of SDS and MPD can promote membrane protein folding and can even be used in membrane protein crystallization. We will investigate the mechanism of how MPD modulates the properties of SDS, and use this information to expand the applications of this technology. Overall, the proposed research program describes our plans to expand on these new methodologies. The focus is to develop and characterize these systems in order to understand them at the molecular level, as well as to explore how they can be applied to existing and novel applications.