Ultraviolet resonance Raman and fluorescence studies of folded and unfolded conformations of the membrane protein OmpA.

摘要

This dissertation focuses on the folding dynamics of a bacterial membrane protein, Outer Membrane Protein A (OmpA), using fluorescence and Ultraviolet resonance Raman spectroscopy. Our model beta-barrel membrane protein, OmpA, contains five native anchoring Tryptophan residues. The spectroscopic properties of trp residues are highly sensitive to the local environment, making it an ideal probe for membrane protein folding studies. Utilizing trp fluorescence, refolding studies were performed on single trp mutants of OmpA to determine the thermodynamic stability of these trp mutants. The important noncovalent interactions that promote stability in OmpA are pairwise aromatic interactions and hydrogen bonds with the N1H moiety of trp. Refolding studies were also performed on truncated single-trp mutants, in which the soluble domain of the protein was removed. These studies resulted in increased stability relative to the full-length protein and suggest the absence of the soluble domain may destabilize the unfolded transmembrane domain.;Ultraviolet resonance Raman spectroscopy (UVRR) is a powerful vibrational technique that can selectively probe different biological chromophores depending on excitation wavelength. Excitation wavelength dependence studies were performed on OmpA using wavelengths from 206.5 nm--236.5 nm. This study determined an optimal excitation wavelength of 228-nm to selectively enhance signal from trp residues in OmpA. Additionally, UVRR was used to monitor changes in trp environmental hydrophobicity, hydrogen bonding, and dihedral torsion angle in different conformations of OmpA. The first UVRR spectra were collected of OmpA in a highly scattering environment and show differences in folded and unfolded conformations of the protein, showing the applicability of this technique to study membrane protein folding. UVRR spectra were collected of trp mutants of OmpA at different time points in the folding/insertion process to determine the types of noncovalent interactions with trp residues, and the folding timescales these interactions occur. Our results indicate noncovalent interactions start to form within the first 20 minutes after initiation of folding into DMPC vesicles and continue to show subtle changes over the course of the folding process. Additionally, there is evidence for interactions between trp residues and lipids, inter-residue hydrogen bonding, and amino-aromatic interactions.