Investigating Ion Channels using Chemical Synthesis

Abstract

Chemical synthesis is a powerful method for precise modification of the structural and electronic properties of proteins. The difficulties in the synthesis and purification of peptides containing transmembrane segments have presented obstacles to the chemical synthesis of integral membrane proteins. We will present a modular strategy for the semi-synthesis of integral membrane proteins in which solid phase peptide synthesis is limited to the region of interest, while the rest of the protein is obtained by recombinant means. This modular strategy considerably simplifies the synthesis and purification steps that have previously hindered the chemical synthesis of integral membrane proteins. We will discuss a sandwich-intein fusion strategy and a sumo-fusion and proteolysis approach for obtaining the membrane spanning peptides required for the semi-synthesis. We will demonstrate the feasibility of the modular approach by the semi-synthesis of the K+ channel, KcsA and the non-selective cation channel NaK. The use of chemical synthesis in functional investigations of the KcsA and the NaK channels will also be presented.