Fluorescence and energy transfer studies of membrane protein folding
- Author(s): Kang, Guipeun
- et al.
Approximately 30 % of human genes encode for membrane proteins. Membrane proteins play important roles in cells as ion pumps, ligand receptors, and ion channels, and are targets for approximately 60 % of all therapeutic drugs. Despite their relevance in biology and biochemistry, membrane proteins account for only 1 % of the total number of reported protein structures (RCSB Protein Data Bank), and only 16 % of all protein folding literature is related to membrane protein folding (literature search from 1990 - - 2015, Web of Science). This dissertation presents spectroscopic studies of the in vitro folding mechanisms of outer membrane protein A (OmpA). Several optical tools are utilized, including circular dichroism (CD), tryptophan fluorescence, Förster resonance energy transfer (FRET), and ultraviolet resonance Raman (UVRR) spectroscopy. An improved method to determine free energies of unfolding of OmpA based on spectral decomposition is presented. Dynamics of OmpA folding in synthetic lipid bilayers of small unilamellar vesicles (SUVs) are investigated through studies of secondary and tertiary structures. CD and FRET data indicate that secondary and tertiary structures are formed within the first hour of folding, and strand extension and equilibration continues on a longer timescale. UVRR data complement the CD and FRET results, and reveal evolution of molecular interactions during folding. In particular, a tryptophan residue in the extra-vesicle portion of the pore (position 129) displays unusually intense Raman activity in the hydrogen-out-of-plane (HOOP) region. The increase in HOOP intensity is hypothesized to reflect perturbation of the indole ring electron density because of a nearby charged residue or hydroxyl group on neighboring threonine residue. More likely, hydrogen bonding of [pi] electrons on tryptophan with hydroxyl group contributes to the overall stability in addition to hydrophobic contacts by neighboring hydrophobic residues. A relatively new folding environment of nanodiscs is also explored. Preliminary FRET and UVRR data show that OmpA folds and inserts into nanodiscs. Collectively, these measurements elucidate changes in secondary and tertiary structures as well as molecular interactions of tryptophan residues during membrane protein folding