Molecular self-assembly and interactions in solutions of membrane proteins and surfactants

Membrane proteins are amphiphilic proteins that are associated with biological membranes. They regulate critical functions between the cells and their surroundings, yet the relationship between structure and functionality for most of these proteins is still elusive. Integral membrane proteins often span the entire bilayers, and they are characterized by a hydrophobic domain that rests in the membrane and hydrophilic regions on either side of the membrane. These proteins are removed from their native membranes for purification and characterization, and surfactants are typically used to solubilize the hydrophobic portion of the molecule. Unfortunately, membrane proteins often exhibit poor stability when solubilized in surfactant solutions and they are very difficult to crystallize. The goal of this dissertation is to contribute to the understanding of how the self-assembly of surfactants in solution affects the stability of solubilized membrane proteins. Liquid-liquid phase boundaries in surfactant solutions have been suggested to have a prominent role in promoting the crystallization of protein-surfactant complexes. Medium-chain alkyl monoglucosides are highly soluble nonionic surfactants that are widely used for the solubilization of membrane proteins. In combinations with more hydrophobic surfactants or water-soluble polymers such as polyethylene glycol (PEG), solutions of these alkyl monoglucosides exhibit miscibility gaps that can be shifted rationally in the temperature and concentration windows. Isothermal titration calorimetry is used to quantify the effect of PEG on the micellization properties of the alkyl monoglucosides, whereas small-angle neutron scattering gives insight into the microstructure of the mixtures near liquid-liquid phase boundaries. Analysis of the scattering profiles using indirect Fourier transformation methods reveals that the role of PEG is to drastically change the strength and range of intermicellar interactions with minimal impact on the geometry of the micelles. These observations confirm the role of the surfactant phase boundary on tuning attractive micellar interactions, and can be related to current protein crystallization strategies. Understanding how surfactants bind to membrane proteins and affect their stability is essential for the manipulation of these proteins outside native membranes. Contrast variation studies by analytical ultracentrifugation and small-angle neutron scattering enable measurement of the composition of the protein-surfactant complexes and determination of the thickness of the surfactant shell bound to the protein. When bacteriorhodopsin is solubilized in solutions of alkyl polyglucosides, the surfactant layer around the protein has a thickness equal to a single amphiphile molecule or larger. The thickness of the surfactant shell increases with increasing surfactant length, and it is generally unrelated to the aggregation number of the micelles even for small and predominantly hydrophobic membrane proteins. Studies of bacteriorhodopsin activity by absorption spectroscopy show that the surfactant arrangement as a single layer directly correlates with a limited stability of the protein over time. A similar connection between surfactant binding and protein stability is observed when bacteriorhodopsin is illuminated and active in pumping protons. These results are useful to guide the choice of surfactant solutions for optimal solubilization of membrane proteins, which is the key to increasing success rates in crystallization and functional studies of these proteins.