Understanding structure and function of membrane protein transporters

Abstract: Membrane protein transporters are important proteins in the cell, as they maintain different solutes and metabolites at a stable concentration. Numerous diseases can be linked to malfunctioning transporters, which makes them interesting pharmaceutical targets. To learn more how these proteins are regulated both functional and structural studies are needed. Membrane proteins are challenging since it is not easy to produce large amounts of these proteins and they readily precipitate due to their hydrophobic nature. In this thesis the function or structure of three different membrane protein transporters were studied; Melibiose permease (MelB), a sugar co-transporter from Escherichia coli, a potassium channel (Kch) from Escherichia coli and a divalent ion transporter from Thermotoga maritima (CorA). Studies of two-dimensional crystals from MelB with Cryo electron microscopy resulted in a three-dimensional structure to 10 Å. The structure revealed an asymmetrical cone shaped molecule that clearly was closed in one end and open in the other. The overall structure resembled that of the Na+/H+ antiporter (NhaA) more than that of the lactose permease (LacY). Preliminary results from crystals without substrate show structural differences in the projection map. Also crystals without substrate were obtained, which preliminary show structural differences in the projection map. Single particle studies of Kch indicated that the majority of tetramers of the protein probably dimerize in a way that can explain why the protein has been difficult to reconstitute into liposomes. Functional studies of CorA from Thermotoga maritima demonstrated that the substrate may be Co2+, and thus Mg2+ may not be the primary substrate of all CorA proteins as previously anticipated. Furthermore fluorescence measurements of tryptophan quenching indicated that there are two binding sites for Co2+ with different affinities (Kd values was 28 ?M and 750 ?M). During these studies methods to obtain stable protein were investigated. A small-scale approach for a buffer screen with analytical size exclusion chromatography was developed. With this method a protein, that previously precipitated during ultrafiltration was possible to concentrate to amounts suitable for crystallization trials. Also, the activity of TEV protease was investigated in different detergents. This protease is commonly used to remove affinity tags from proteins. Our results show that the choice of the detergent affects the accessibility of the membrane protein cleavage site and not the activity of the TEV protease. Taken together; the results presented in this thesis deals with problems of stability of purified membrane proteins and protease activity it detergent solutions. Structures of two membrane proteins (MelB and Kch) were determined with two different electron microscopy methods, and the substrate specificity of CorA was explored and revised