The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator

Abstract

Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed.

Covalently Linking Oligomerization-Impaired GlpF Protomers Does Not Completely Re-establish Wild-Type Channel Activity

“…The AQP translocation pore is defined in the monomer (Figure 1), and thus, AQPs are facultative oligomers [6]. AQPs assemble into stable homotetramers in vivo and in vitro, resulting in formation of an additional fifth pore in the center of the tetramer [7,8,9,10,11,12]. The formation of this additional pore, which possibly allows the flux of gaseous substrates such as CO 2 and NO across the lipid bilayer, is suggested to be a driving force for AQP tetramerization [13,14].…”