Synthesis, biosynthesis, and characterization of transmembrane domains of a G protein-coupled receptor.

Abstract

Peptide fragments have been widely used in biophysical studies on specific regions of integral membrane proteins. Because of their inherent insoluble nature and tendency to aggregate the preparation of such model peptides is challenging. We have developed synthetic and biosynthetic approaches to prepare peptides containing single and multiple domains of a G protein-coupled receptor. Both the synthetic and biosynthetic products can be isolated by reversed-phase high-performance liquid chromatography to near homogeneity. The biosynthetic product, a fusion protein, is processed by CNBr cleavage to yield the target peptide in various isotopic forms. The final peptides are studied by circular dichroism spectroscopy to determine their secondary structure under a variety of conditions.