Hydrophobic Mismatch Modulates Stability and Plasticity of Human Mitochondrial VDAC2.

The human mitochondrial outer membrane protein voltage-dependent anion channel isoform 2 (hVDAC2) is a ß-barrel metabolite flux channel that is indispensable for cell survival. It is well established that physical forces imposed on a transmembrane protein by its surrounding lipid environment decide protein structure and stability. Yet, how the mitochondrial membrane and protein-lipid interplay together regulate hVDAC2 stability is unknown. Here, we combine experimental biophysical investigations of protein stability with all-atom molecular dynamics simulations to study the effect of the most abundant mitochondrial phosphocholine (PC) lipids on hVDAC2. We demonstrate experimentally that increasing the PC lipid acyl chain length from diC14:0 to diC18:0-PC has a nonlinear effect on the ß-barrel. We show that protein stability is highest in diC16:0-PC, which exhibits a negative mismatch with the hVDAC2 barrel. Our simulations also reveal that structural rigidity of hVDAC2 is highest under optimal negative mismatch provided by diC16:0-PC bilayers. Further, we validate our observations by altering the physical properties of PC membranes indirectly using cholesterol. We propose that VDAC plasticity and stability in the mitochondrial outer membrane are modulated by physical properties of the bilayer.

Salvaging the Thermodynamic Destabilization of Interface Histidine in Transmembrane ß-Barrels.

The ability of histidine to participate in a wide range of stabilizing polar interactions preferentially populates this residue in functionally important sites of proteins. Histidine possesses an amphiphilic and electrostatic nature that is essential for amino acids residing at membrane interfaces. However, the frequency of occurrence of histidine at membrane interfaces, particularly transmembrane ß-barrels, is lower than those of other aromatic residues. Here, we carry out comprehensive energetic measurements using equilibrium folding of the outer membrane enzyme PagP to address the contribution of a C-terminal interface histidine to barrel stability. We show that placing histidine at the C-terminus universally destabilizes PagP by 4.0-8.0 kcal mol-1 irrespective of the neighboring residue. Spectroscopic and electrophoretic measurements indicate that the altered stability may arise from a loss of barrel compaction. Isoleucine, methionine, and valine salvage this destabilization marginally (in addition to tyrosine, which shows an exceptionally high folding free energy value), when placed at the penultimate position, at the expense of an altered folding pathway. Double-mutant cycle analysis indicates that the coupling energy between the terminal and penultimate residues in PagP-X160H161 increases when the level of intrinsic destabilization by the terminal H161 is high. Our observations that neighboring residues cannot salvage the energetic destabilization of histidine may explain why histidine is less abundant at membrane interfaces.

Energetics of side-chain partitioning of ß-signal residues in unassisted folding of a transmembrane ß-barrel protein.

The free energy of water-to-interface amino acid partitioning is a major contributing factor in membrane protein folding and stability. The interface residues at the C terminus of transmembrane ß-barrels form the ß-signal motif required for assisted ß-barrel assembly in vivo but are believed to be less important for ß-barrel assembly in vitro Here, we experimentally measured the thermodynamic contribution of all 20 amino acids at the ß-signal motif to the unassisted folding of the model ß-barrel protein PagP. We obtained the partitioning free energy for all 20 amino acids at the lipid-facing interface (ΔΔG0w,i(φ)) and the protein-facing interface (ΔΔG0w,i(π)) residues and found that hydrophobic amino acids are most favorably transferred to the lipid-facing interface, whereas charged and polar groups display the highest partitioning energy. Furthermore, the change in non-polar surface area correlated directly with the partitioning free energy for the lipid-facing residue and inversely with the protein-facing residue. We also demonstrate that the interface residues of the ß-signal motif are vital for in vitro barrel assembly, because they exhibit a side chain-specific energetic contribution determined by the change in nonpolar accessible surface. We further establish that folding cooperativity and hydrophobic collapse are balanced at the membrane interface for optimal stability of the PagP ß-barrel scaffold. We conclude that the PagP C-terminal ß-signal motif influences the folding cooperativity and stability of the folded ß-barrel and that the thermodynamic contributions of the lipid- and protein-facing residues in the transmembrane protein ß-signal motif depend on the nature of the amino acid side chain.

Differential contribution of tryptophans to the folding and stability of the attachment invasion locus transmembrane ß-barrel from Yersinia pestis.

Attachment invasion locus (Ail) protein of Yersinia pestis is a crucial outer membrane protein for host invasion and determines bacterial survival within the host. Despite its importance in pathogenicity, surprisingly little is known on Ail biophysical properties. We investigate the contribution of micelle concentrations and interface tryptophans on the Ail ß-barrel refolding and unfolding processes. Our results reveal that barrel folding is surprisingly independent of micelle amounts, but proceeds through an on-pathway intermediate that requires the interface W42 for cooperative barrel refolding. On the contrary, the unfolding event is strongly controlled by absolute micelle concentrations. We find that upon Trp → Phe substitution, protein stabilities follow the order W149F>WT>W42F for the refolding, and W42F>WT>W149F for unfolding. W42 confers cooperativity in barrel folding, and W149 clamps the post-folded barrel structure to its micelle environment. Our analyses reveal, for the first time, that interface tryptophan mutation can indeed render greater ß-barrel stability. Furthermore, hysteresis in Ail stems from differential barrel-detergent interaction strengths in a micelle concentration-dependent manner, largely mediated by W149. The kinetically stabilized Ail ß-barrel has strategically positioned tryptophans to balance efficient refolding and subsequent ß-barrel stability, and may be evolutionarily chosen for optimal functioning of Ail during Yersinia pathogenesis.