Folding of a beta-barrel membrane protein into nanodiscs

Membrane proteins constitute a significant fraction of cellular proteins due to their vast diversity in function. However, studies of their native structures, folding mechanisms, and dynamics remain a challenge, in part because of their inherent insolubility. One goal of this project is to enhance the tools to study membrane proteins. Small unilamellar vesicles (SUVs) have been used as a bilayer mimetic to investigate membrane protein structures, dynamics, and folding mechanisms. UV resonance Raman spectroscopy (UVRR) has emerged as a powerful technique for probing structures and dynamics of biomolecules. UVRR is particularly valuable for membrane proteins because of the selective enhancement of signal from key aromatic residues and the ability to probe interactions between membrane protein and the lipid. We present practical considerations and guidelines for UVRR data acquisition, including a detailed description of a typical laser setup as well as the process to analyze the tryptophan vibrational modes of a model β-barrel membrane protein, OmpA, unfolded and folded in SUVs. An additional need in studies of membrane protein folding is alternate membrane mimics. Nanodiscs (NDs) are an excellent bilayer alternative to SUVs because of their experimental benefits including homogeneity, optical clarity, low light scattering, and enhanced stability. We combine SDS-PAGE mobility studies with fluorescence, circular dichroism, and UVRR spectroscopy to confirm and characterize the folding of OmpA into NDs. Our studies show similar secondary and tertiary structures in both SUVs and NDs as well as efficient folding yields greater than 88% in both bilayers. The folding of OmpA into NDs was slower compared to in SUVs, and this difference can be attributed to the different bilayer characteristics. Insights into the folding mechanism of OmpA were gained via bimolecular fluorescence quenching with acrylamide quencher. Stern-Volmer analysis utilizing the sphere-of-action model probe