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 haveinvestigated 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 perturbingthe 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.
Highlights
Voltage dependent anion channels (VDACs), which are eukaryotic mitochondrial outer membrane proteins [1], conduct metabolites and ionsincluding ATP and NADH [1,2]between the intermembrane space and cytosol
Using the intrinsic fluorescence of the four Trp residues of human VDAC isoform 2 (hVDAC-2), we acquired data for barrel unfolding as a function of time.Surprisingly, we find that both hVDAC-2 Wild type hVDAC-2 (WT) and C0 do not undergo complete unfolding even after h incubation in 7.5 M urea, at the lowest lauryldimethylamine oxide (LDAO) concentrations used in this study (Figure 5).This is unexpected, when we consider that the thermodynamic stability of hVDAC-2 in LDAO micelles is considerably lower (1.8 – 4.5 kcal/mol [24] than soluble proteins of comparable molecular weights (8– kcal/mol [54,55,56,57]).While both urea and GdnHCl have been employed in equilibrium thermodynamics studies, a few bacterial transmembrane b-barrels are known to exhibit a differential response to urea as a denaturant
Of the three human VDAC isoforms, hVDAC-2 and hVDAC-3 are enriched with cysteine residues. hVDAC-1 cysteines are known to exist in the reduced state and are not required for barrel functioning [22]
Summary
Voltage dependent anion channels (VDACs), which are eukaryotic mitochondrial outer membrane proteins [1], conduct metabolites and ionsincluding ATP and NADH [1,2]between the intermembrane space and cytosol. In addition to the controlled transport of vital metabolites in the cell, most interestingly, VDACs are deciding elements of mitochondria-mediated apoptosis [4,11,12], and have been implicated in several neurodegenerative diseases [5,13]and cancer [5,14,15].Of key significance is the observation that human VDAC isoform 2 (hVDAC-2) may possess antagonistic functions compared to hVDAC-1 [4,11]. The presence of all three isoforms is not essential for cell survival; surprisingly, unlike isoforms 1 and 3, it was observed that hVDAC22/2 mice died at the embryonic stage and could not be rescued by the over-expression of VDAC-1 [5,11,17,18,19].This led to the serendipitous discovery that hVDAC-2 possesses anti-apoptotic property, likely serves as a specific inhibitor of BAK-dependent mitochondria-mediated apoptosis, and is indispensable for cell survival [5,11,17,18,19]
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