Influence of protein-micelle ratios and cysteine residues on the kinetic stability and unfolding rates of human mitochondrial VDAC-2.
Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane β-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 β-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 β-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.
Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh, India.
Svetlana Rajkumar
SR
Maurya
Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh, India.
Radhakrishnan
R
Mahalakshmi
D003545:Cysteine; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D020033:Protein Isoforms; D055550:Protein Stability; D058767:Protein Unfolding; D013816:Thermodynamics; D050996:Voltage-Dependent Anion Channel 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins; D008823:Micelles; D024101:Mitochondrial Proteins; D020033:Protein Isoforms; D050996:Voltage-Dependent Anion Channel 2; D003545:Cysteine