Protein and Environmental Determinants of OMP Fate in Liposomes

Abstract

Despite the vast importance of outer membrane proteins (OMPs), OMPs are understudied because they must first be folded into a liposome or other membrane mimetic.1 There is no ideal folding condition that works for all OMPs, so empirical screens are used to determine the best conditions for a given OMP to fold.2 We aim to analyze the fates of various OMPs (fold, aggregate, or lipid-associate without folding) in a variety of conditions to understand the protein (particularly extracellular loop), lipid, and buffer characteristics that determine the proteins’ fate. Theoretical understanding of folding determinants would significantly decrease the time needed to find a folding system for any particular OMP, which could expedite in vitro work on membrane proteins. Four OMPs were chosen for study: OmpA, OmpW, Opa60 and OmpX. OmpA and OmpX are promiscuous folders, while Opa60 and OmpW are difficult folders.2,3 All four are eight stranded β-barrels, with extracellular loops of varying size, pI, and hydrophobicity. The OMPs were overexpressed to inclusion bodies and purified using centrifugation and wash steps, with final purity demonstrated by SDS-PAGE. OMPs were folded into liposomes (SUVs and LUVs) and protein aggregate was separated from folded and lipid-associated protein via ultra-centrifugation.3 Folded and unfolded protein in each fraction was determined by SDS-PAGE and gel densiometry, as fully folded β-barrels migrate differently than lipid-associated protein through a polyacrylamide gel. Results for the four OMPs at various pH, lipid chain length, and salt concentrations are presented. 1. LK Tamm, H Hong, and B Liang. (2004) Biochim Biophys Acta, 1666, 250. 2. NK Burgess, TP Dao, AM Stanley, KG Fleming. (2008) J Biol Chem, 283, 26748. 3. AH Dewald, JC Hodges, L Columbus. (2011) Biophys J, 100, 2131.