FRET Investigation of Membrane Protein Folding: Evolution of Tertiary Structure of Soluble and Transmembrane Domains during Folding into Synthetic Bilayers

Abstract

This paper describes an investigation of the folding mechanisms of an integral membrane protein. A key goal is to determine the role of the soluble domain during the insertion of a transmembrane domain into synthetic bilayers. Towards this end, we report Forster resonance energy transfer (FRET) efficiencies between donor (tryptophan) and acceptor (1,5-IAEDANS) pairs that are located on the transmembrane and soluble domains of outer membrane protein A (OmpA). The FRET efficiencies are correlated to the evolution of distances and tertiary structure under the assumption of orientational averaging. Analysis of the kinetics reveals that the full-length protein, which contains both soluble and transmembrane domains, displays slower folding rates compared to the truncated variant, which is comprised of the transmembrane domain only. This difference in rates may reflect an increase in the number of kinetic traps during folding, or indicate alternate pathways. These measurements of the formation of tertiary structure and the role of a soluble domain on the kinetics of folding may aid in the elucidation of the mechanisms of membrane protein folding and dynamics.