Stability of the Glycerol Facilitator in Detergent Solutions

Abstract

Understanding membrane protein folding and stability is required for a molecular explanation of function and for the development of interventions in membrane protein folding diseases. Stable aqueous detergent solutions of the Escherichia coli glycerol facilitator in its native oligomeric state have been difficult to prepare as the protein readily unfolds and forms nonspecific aggregates. Here, we report a study of the structure and stability of the glycerol facilitator in several detergent solutions by Blue Native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD), and fluorescence. Protein tetramers were prepared in neutral dodecyl maltoside (DDM) and in zwitterionic lysomyristoylphosphatidylcholine (LMPC) detergent solutions that are stable during SDS-PAGE. Thermal unfolding experiments show that the protein is more stable in LMPC than in DDM. Tertiary structure unfolds before quaternary and some secondary structure in LMPC, whereas unfolding is more cooperative in DDM. The high stability of the protein in DDM is evident from the unfolding half-life of 8 days in 8 M urea, suggesting that hydrophobic interactions contribute to the stability. The protein unfolds readily in LMPC below pH 6, whereas the tetramer remains intact at pH 4 in DDM. At pH 4 in DDM, the protein is more sensitive than at neutral pH to unfolding by SDS and the effect is reversible. At pH 3 in DDM, the tetramer unfolds, losing its tertiary structure but retaining native helical structure which melts at significantly lower temperatures than in the native tetramer. The glycerol facilitator prepared in SDS is mainly monomeric and has 10% less R-helix than the native protein. CD suggests that it forms a condensed structure with non-native tertiary contacts highly similar to the state observed in LMPC at low pH. The implications of the results for in vitro and in vivo folding of the protein are discussed.

Anionic Lipids Modulate the Activity of the Aquaglyceroporin GlpF

“…The amount of liposome-integrated protein was determined via a semi-native SDS-PAGE analysis. In this analysis, no additional SDS is added to the sample buffer and the native tetrameric oligomerization state of GlpF is preserved (23,30). For subsequent analysis, proteoliposomes were incubated in SDS-free sample buffer (50 mM Tris-HCl (pH 6.8; Roth, Karlsruhe, Germany), 10% (v/v) glycerol (AppliChem, Darmstadt, Germany), and 0.04% (w/v) Bromphenol blue (Sigma-Aldrich, Munich, Germany) for 15 min at room temperature and separated on a 10% SDS-PAGE gel.…”

“…During such an analysis, the tetrameric state of GlpF remains intact (23,24,30). This remarkable feature of GlpF was utilized to assess the stability of the tetrameric GlpF complex in different lipid environments, as determined by SDS-induced unfolding of the tetramer to monomeric, dimeric, and potentially trimeric GlpF (23).…”

Conformational Dynamics of a Membrane Transport Protein Probed by H/D Exchange and Covalent Labeling: The Glycerol Facilitator

“…Each of the 33.9-kDa subunits forms one pore, for a total of four transmembrane channels per tetramer. 14,15,[19][20][21] This quaternary structure can be maintained after solubilization in suitable detergents. [19][20][21] GF-mediated permeation of water through the membrane proceeds roughly twice as fast as that of glycerol.…”

Expression, purification, and preliminary characterization of human presenilin-2

“…This is in agreement with the irreversibility of structural transformations of hisPS2 as observed from the temperature reverse scan, the deconvoluted structure composition at different temperatures, and the detection of SDS-resistant aggregates. The presence of 22% helix at 98°C was probably due to the extreme stability of the detergent-bound helix, which prevented it from unfolding − a phenomenon also observed for other membrane proteins [79,80]. Cholesterol significantly increased the thermal stability of hisPS2, which provides evidence that cholesterol acts as a natural ligand.…”