Building An Artificial Membrane Protein: Design, Expression and Characterization in Micelles and Lipid Vesicles

Abstract

High efficiencies and catalytic activities of natural oxido-reductases keep inspiring development of new enzymes with the goal to produce better catalysts, novel drug therapies, bioelectronics devices, bioremediation and alternative energy sources. However, the immense complexity of natural proteins and their very evident resistance and fragility to modification prevents scientists from deciphering what part of the protein structure is essential for reproducing the catalytic function itself apart from the many other demands made on a natural protein within a cell. To overcome this obstacle, we are designing general structural platforms (maquettes) that accommodate variety of oxido-reductase functions, including light energy harvesting, photochemical charge separation, oxygen transport, and oxidative metabolism. They also serve as models for understanding the fundamental properties of enzyme activity, stability, and folding. Here we will present a transmembrane maquette designed to form electron transfer chain across a lipid bilayer. This artificial membrane maquette contains four membrane spanning α-helices that are linked into a single chain. It has been expressed in high yields in inclusion bodies using E. Coli, purified and refolded in charged and uncharged detergent micelles, as well as in lipid vesicles. Circular dichroism studies revealed 70% α-helical nature in SDS and no melting in high temperatures and common denaturants, indicating its strong structural stability. We will present maquette assembly, stability, cofactor binding and redox characteristics in different membrane environments and how they compare to natural proteins.