The effects of a protein osmolyte, detergent headgroup, and detergent chain length on the stability of the integral membrane protein glycerol facilitator.

摘要

One of the main impediments to membrane protein research is the lower stability of the proteins following their removal from the lipid bilayer. Osmolytes are naturally-occurring molecules used by a wide variety of organisms to stabilize proteins under conditions of high salinity, high hydrostatic pressure, desiccation, and high and low temperatures. Osmolytes have also been shown to protect proteins from chemical denaturants such as urea in kidney cells. The results of studies to determine the effects of the osmolyte trimethylamine N-oxide (TMAO) on the stability of the E. coli integral membrane protein glycerol facilitator (GlpF) are presented. TMAO promoted the association of the normally tetrameric alpha-helical protein into an octameric species in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC). Both the tetramer and octamer are significantly more heat stable in the presence of TMAO. The osmolyte also stabilizes the protein against denaturation by sodium dodecyl sulphate (SDS). A concentration-dependence of TMAO in stabilizing against SDS denaturation was also observed in all detergents, with high levels of octamer in DDM only being found at high TMAO concentrations. Protein structure was monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS). The latter technique was applied to a membrane protein for the first time. It is also found that the protein is more stable in detergents with the phosphatidylcholine head group (LLPC and LMPC), and the least stable in TDM. These results may contribute to improved methodology for studying membrane proteins and a better understanding of membrane protein structure, folding and dynamics.