Lipid bilayer composition modulates the unfolding free energy of a knotted α-helical membrane protein

α-Helical membrane proteins have eluded investigation of their thermodynamic stability in lipid bilayers. Reversible denaturation curves have enabled some headway in determining unfolding free energies. However, these parameters have been limited to detergent micelles or lipid bicelles, which do not possess the same mechanical properties as lipid bilayers that comprise the basis of natural membranes. We establish reversible unfolding of the membrane transporter LeuT in lipid bilayers, enabling the comparison of apparent unfolding free energies in different lipid compositions. LeuT is a bacterial ortholog of neurotransmitter transporters and contains a knot within its 12-transmembrane helical structure. Urea is used as a denaturant for LeuT in proteoliposomes, resulting in the loss of up to 30% helical structure depending upon the lipid bilayer composition. Urea unfolding of LeuT in liposomes is reversible, with refolding in the bilayer recovering the original helical structure and transport activity. A linear dependence of the unfolding free energy on urea concentration enables the free energy to be extrapolated to zero denaturant. Increasing lipid headgroup charge or chain lateral pressure increases the thermodynamic stability of LeuT. The mechanical and charge properties of the bilayer also affect the ability of urea to denature the protein. Thus, we not only gain insight to the long–sought-after thermodynamic stability of an α-helical protein in a lipid bilayer but also provide a basis for studies of the folding of knotted proteins in a membrane environment.

The intersubunit lock-and-key motif in human glutathione transferase A1-1: role of the key residues Met51 and Phe52 in function and dimer stability

The dimeric structure of certain cytosolic GSTs (glutathione S-transferases) is stabilized by a hydrophobic lock-and-key motif at their subunit interface. In hGSTA1-1 (human class Alpha GST with two type-1 subunits), the key consists of two residues, Met51 and Phe52, that fit into a hydrophobic cavity (lock) in the adjacent subunit. SEC (size-exclusion chromatography)–HPLC, far-UV CD and tryptophan fluorescence of the M51A and M51A/F52S mutants indicated the non-disruptive nature of these mutations on the global structure. While the M51A mutant retained 80% of wild-type activity, the activity of the M51A/F52S was markedly diminished, indicating the importance of Phe52 in maintaining the correct conformation at the active site. The M51A and M51A/F52S mutations altered the binding of ANS (8-anilinonaphthalene-l-sulphonic acid) at the H-site by destabilizing helix 9 in the C-terminal region. Data from urea unfolding studies show that the dimer is destabilized by both mutations and that the dimer dissociates to aggregation-prone monomers at low urea concentrations before global unfolding. Although not essential for the assembly of the dimeric structure of hGSTA1-1, both Met51 and Phe52 in the intersubunit lock-and-key motif play important structural roles in maintaining the catalytic and ligandin functions and stability of the GST dimer.