title: Cysteine residues impact the stability and micelle interaction dynamics of the human mitochondrial β-barrel anion channel hVDAC-2.
abstract: The anti-apoptotic 19-stranded transmembrane human voltage dependent anion channel isoform 2 (hVDAC-2) β-barrel stability is crucial for anion transport in mitochondria. The role of the unusually high number of cysteine residues in this isoform is poorly understood. Using a Cys-less construct of hVDAC-2, we have investigated the contribution of cysteines to channel function, barrel stability and its influence on the strength of protein-micelle interactions. We observe that despite the overall preservation in barrel structure upon cysteine mutation, subtle local variations in the mode of interaction of the barrel with its refolded micellar environment arise, which may manifest itself in the channel activity of both the proteins.Fluorescence measurements of the Trp residues in hVDAC-2 point to possible differences in the association of the barrel with lauryldimethylamine oxide (LDAO) micelles. Upon replacement of cysteines in hVDAC-2, our data suggests greater barrel rigidity by way of intra-protein interactions. This, in turn, lowers the equilibrium barrel thermodynamic parameters in LDAOby perturbing the stability of the protein-micelle complex. In addition to this, we also find a difference in the cooperativity of unfolding upon increasing the LDAO concentration, implying the importance of micelle concentration and micelle-protein ratios on the stability of this barrel. Our results indicate that the nine cysteine residues of hVDAC-2 are the key in establishing strong(er) barrel interactions with its environment and also impart additional malleability to the barrel scaffold.
authors.affiliation: Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh, India.
authors.forename: Svetlana Rajkumar
authors.initials: SR
authors.lastname: Maurya
authors.affiliation: Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh, India.
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Mahalakshmi
mesh_terms: D019943:Amino Acid Substitution; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004123:Dimethylamines / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008823:Micelles; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D050996:Voltage-Dependent Anion Channel 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004123:Dimethylamines; D008823:Micelles; D020033:Protein Isoforms; D011994:Recombinant Proteins; C494765:VDAC2 protein, human; D050996:Voltage-Dependent Anion Channel 2; C014518:dodecyldimethylamine oxide; D014364:Tryptophan; D003545:Cysteine