Influence of protein-micelle ratios and cysteine residues on the kinetic stability and unfolding rates of human mitochondrial VDAC-2.

ABSTRACT: Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane ?-barrels is critical to gain an in-depth understanding of membrane protein behavior. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2), one of the key anti-apoptotic eukaryotic ?-barrel proteins, is of paramount importance, owing to its indispensable role in cell survival. We demonstrate here that the stability of hVDAC-2 bears a strong kinetic contribution that is dependent on the absolute micellar concentration used for barrel folding. The refolding efficiency and ensuing stability is sensitive to the lipid-to-protein (LPR) ratio, and displays a non-linear relationship, with both low and high micellar amounts being detrimental to hVDAC-2 structure. Unfolding and aggregation process are sequential events and show strong temperature dependence. We demonstrate that an optimal lipid-to-protein ratio of 2600?1 - 13,000?1 offers the highest protection against thermal denaturation. Activation energies derived only for lower LPRs are ?17 kcal mol(-1) for full-length hVDAC-2 and ?23 kcal mol(-1) for the Cys-less mutant, suggesting that the nine cysteine residues of hVDAC-2 impart additional malleability to the barrel scaffold. Our studies reveal that cysteine residues play a key role in the kinetic stability of the protein, determine barrel rigidity and thereby give rise to strong micellar association of hVDAC-2. Non-linearity of the Arrhenius plot at high LPRs coupled with observation of protein aggregation upon thermal denaturation indicates that contributions from both kinetic and thermodynamic components stabilize the 19-stranded ?-barrel. Lipid-protein interaction and the linked kinetic contribution to free energy of the folded protein are together expected to play a key role in hVDAC-2 recycling and the functional switch at the onset of apoptosis.

SUBMITTER: Maurya SR 

PROVIDER: S-EPMC3907894 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature