A new class of amphiphiles bearing rigid hydrophobic groups for solubilization and stabilization of membrane proteins.

Integral membrane proteins (IMPs) are crucial cellular components, mediating the transfer of material and signals between the environment and the cytoplasm, or between different cellular compartments. Structural and functional analysis of IMPs is important; more than half of current pharmaceutical agents target proteins in this class. IMP characterization is often challenging, and sometimes impossible, because of difficulties associated with handling these macromolecules. IMPs in the native state display large hydrophobic surfaces, which are not compatible with an aqueous environment; therefore, detergents are required to extract IMPs from the lipid bilayer and to maintain the native state of the protein in solution. Nonionic detergents, such as dodecyl-b-d-maltoside (DDM) and octyl-b-d-glucoside (OG), are generally preferred for these applications. Despite the comparatively mild nature of DDM, OG and related detergents, many membrane proteins denature and/or aggregate upon solubilization with these agents. Diverse strategies have been pursued to develop new tools for solubilization of IMPs from membranes and for maintenance of these proteins in a native-like state in aqueous solution. Techniques that are effective for solubilization are not always optimal for stabilization, and vice versa. These efforts have included exploration of novel amphiphilic molecules that depart from traditional detergent architectures. Specifically tailored amphiphiles that facilitate IMP crystallization are particularly noteworthy. Amphiphilic polymers (“amphipols”) and discoidal lipid bilayers stabilized by an amphiphilic protein scaffold (“nanodiscs”) represent highly innovative approaches for stabilizing IMPs in native-like states in aqueous solution. It is not clear, however, whether these approaches can support growth of highquality crystals for diffraction analysis. Furthermore, neither amphipols nor nanodiscs were designed to extract IMPs from biological membranes. Despite considerable progress in the development of new compounds and strategies for membrane protein solubilization and stabilization, new tools are needed, because many IMPs are currently refractory. Given the great variation in structure and physical properties among membrane proteins, it is very unlikely that a single amphiphile or amphiphile family will be optimal for every system, or even most systems, and exploration of new amphiphilic agents is therefore important for membrane protein biochemistry. Herein we report a class of structurally novel amphiphiles that display favorable behavior, relative to traditional detergents such as DDM, toward a diverse set of membrane proteins. The new amphiphiles (Figure 1) all contain a rigid, steroid-based lipophilic group and a di-maltose hydrophilic group. Three of the new compounds are derived from lithocholic acid and are therefore designated “glyco-lithocholate” amphiphiles (GLC-1, GLC-2 and GLC-3); the fourth is derived from diosgenin and designated “glyco-diosgenin” (GDN). Many previously reported amphiphiles based on steroidal skeletons have been derivatives of cholic acid or deoxycholic acid, including members of the widely-used [a] Prof. P. S. Chae, K. H. Cho Department of Bionano Engineering, Hanyang University Ansan, 426-791 (Korea) E-mail : [email protected] [b] Prof. S. H. Gellman Department of Chemistry, University of Wisconsin-Madison 1101 University Avenue, Madison, WI 53706 (USA) E-mail : [email protected] [c] Prof. S. G. F. Rasmussen, A. C. Kruse, A. Manglik, Prof. B. K. Kobilka Molecular and Cellular Physiology, Stanford University Stanford, CA 94305 (USA) E-mail : [email protected] [d] R. R. Rana, Dr. B. Byrne Department of Life Sciences, Imperial College London London, SW7 2AZ (UK) E-mail : [email protected] [e] K. Gotfryd, Prof. U. Gether, Prof. C. J. Loland Department of Neuroscience and Pharmacology University of Copenhagen 2200 Copenhagen (Denmark) E-mail : [email protected] [f] S. Nurva, Prof. L. Guan Department of Cell Physiology and Molecular Biophysics Texas Tech University Health Sciences Center Lubbock, TX 79430 (USA) E-mail : [email protected] Supporting information for this article (including synthesis and characterization of amphiphiles, detergent screening, and stabilization measurements) is available on the WWW under http://dx.doi.org/ 10.1002/chem.201200069.