Design and Characterization of a Single Chain Amphiphilic Maquette for Membrane Insertion and Electron Transfer

Abstract

The ability to design and produce artificial proteins opens up many scientific avenues for producing artificial enzymes, catalysts, novel drug therapies, bioelectronics devices, bioremediation and alternative energy sources. We are designing simplified model proteins, called maquettes, for diverse set of oxidoreductase related functions including electron transfer, catalysis, and charge separation. These maquettes can be built as four-α-helical bundles with different topologies such as homotetramer, homodimer, and single chain (monomer). They can be assembled in aqueous solution (hydrophilic maquettes) or in membranes (amphiphilic and hydrophobic maquettes). Here, we will present the design and characterization of an amphiphilic maquette that has been designed for efficient electron transfer across a lipid bilayer. This maquette contains four membrane-spanning α-helices linked into a single chain. The transmembrane electron transfer is enabled by bis-histidine ligated hemes that are positioned 7-11 Å apart (edge to edge). We have successfully developed methods for expressing this maquette in inclusion bodies using E.coli. Expression in inclusion bodies helps evade problems associated with insolubility and toxicity of the maquette inside the bacterial cells and therefore enables production in high yields. Since we have designed the maquette with very strong alpha-helical propensity, it refolds readily after purification. We will discuss the assembly, heme binding and redox properties of the maquette in detergent micelles and lipid vesicles.