Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

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Biochemical and structural studies of membrane protein usually require their stabilization in solution with detergents. However, purified membrane proteins often show reduced activity and stability in traditional detergents. Lipopeptide detergents (LPDs) are a new class of engineered amphiphiles that from small micelles and mimic the lipid bilayer more closely than traditional detergents. An LPD molecule consists of an α-helical peptide with fatty acyl chains covalently attached to both ends. These molecules self-assemble into cylindrical micelles with a hydrophilic exterior and a hydrophobic interior made up of the fatty acyl chains. Here we present the biophysical and biochemical properties of a model bacterial ABC transporter, MsbA, in different LPD variations. Four types of LPD molecules have been synthesized and have been categorized as original LPD, LPD5Q, LPD2, and LPD4 based on the peptide sequence. Dynamic light scattering, thermal aggregation, and ATPase activity were used to measure the biophysical properties of MsbA in LPDs and in a traditional detergent, n-decyl-β-D-maltoside (DM). The results show that MsbA-LPD particles are monodisperse with small hydrodynamic radii. When compared to DM, MsbA is thermodynamically more stable and has higher catalytic activity in LPDs. Membrane proteins have favorable biophysical properties in LPDs, suggesting that these detergents resemble the native lipid bilayer environment more closely. We also present crystal structures of three LPDs: LPD-12, LPD5Q-14 and LPD- 14. These structures reveal that LPD micelles are highly ordered with varying oligomeric states. The octomeric structure of the LPD-12 micelle is composed of four sets of antiparallel coiled-coil dimers, while the LPD5Q-14 micelle assembles as a nonamer of three trimers each with an “up-up-down” topology. The LPD-14 micelle, on the other hand, is a dodecamer of three tetramers with all helices assuming an antiparallel orientation. Overall, the structures of LPDs show highly ordered detergent micelles that are made up of repeated building blocks. Based on these results, we propose that LPDs can sample multiple conformational states, but the number of accessible conformations is significantly reduced relative to traditional detergents. Our results show that LPDs are an alternative platform for in vitro studies of membrane proteins. Future studies will focus on the crystallization of membrane proteins in LPDs and the further characterization of these complexes.

Biochemical and structural studies of membrane protein usually require their stabilization in solution with detergents. However, purified membrane proteins often show reduced activity and stability in traditional detergents. Lipopeptide detergents (LPDs) are a new class of engineered amphiphiles that from small micelles and mimic the lipid bilayer more closely than traditional detergents. An LPD molecule consists of an α-helical peptide with fatty acyl chains covalently attached to both ends. These molecules self-assemble into cylindrical micelles with a hydrophilic exterior and a hydrophobic interior made up of the fatty acyl chains. Here we present the biophysical and biochemical properties of a model bacterial ABC transporter, MsbA, in different LPD variations. Four types of LPD molecules have been synthesized and have been categorized as original LPD, LPD5Q, LPD2, and LPD4 based on the peptide sequence. Dynamic light scattering, thermal aggregation, and ATPase activity were used to measure the biophysical properties of MsbA in LPDs and in a traditional detergent, n-decyl-β-D-maltoside (DM). The results show that MsbA-LPD particles are monodisperse with small hydrodynamic radii. When compared to DM, MsbA is thermodynamically more stable and has higher catalytic activity in LPDs. Membrane proteins have favorable biophysical properties in LPDs, suggesting that these detergents resemble the native lipid bilayer environment more closely. We also present crystal structures of three LPDs: LPD-12, LPD5Q-14 and LPD- 14. These structures reveal that LPD micelles are highly ordered with varying oligomeric states. The octomeric structure of the LPD-12 micelle is composed of four sets of antiparallel coiled-coil dimers, while the LPD5Q-14 micelle assembles as a nonamer of three trimers each with an “up-up-down” topology. The LPD-14 micelle, on the other hand, is a dodecamer of three tetramers with all helices assuming an antiparallel orientation. Overall, the structures of LPDs show highly ordered detergent micelles that are made up of repeated building blocks. Based on these results, we propose that LPDs can sample multiple conformational states, but the number of accessible conformations is significantly reduced relative to traditional detergents. Our results show that LPDs are an alternative platform for in vitro studies of membrane proteins. Future studies will focus on the crystallization of membrane proteins in LPDs and the further characterization of these complexes.

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