Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh, India.
PLoS One. 2014 Mar 18;9(3):e92183. doi: 10.1371/journal.pone.0092183. eCollection 2014.
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.
人类电压依赖性阴离子通道同工型 2（hVDAC-2）是一种具有 19 个跨膜β桶结构的抗凋亡蛋白，其β桶的稳定性对于线粒体中的阴离子转运至关重要。该同工型中异常高数量的半胱氨酸残基的作用尚未完全了解。本研究使用不含半胱氨酸的 hVDAC-2 构建体，研究了半胱氨酸残基对通道功能、桶稳定性及其对蛋白质-胶束相互作用强度的影响。我们观察到，尽管半胱氨酸突变后桶结构总体上得以保留，但桶与重新折叠的胶束环境相互作用的局部细微变化仍然存在，这可能会影响两种蛋白质的通道活性。hVDAC-2 中色氨酸残基的荧光测量表明，桶与月桂基二甲基氧化胺（LDAO）胶束的结合可能存在差异。在 hVDAC-2 中替换半胱氨酸后，我们的数据表明，通过蛋白质内部相互作用，桶的刚性增加。这反过来又通过扰乱蛋白质-胶束复合物的稳定性，降低了 LDAO 中桶的平衡热力学参数。除此之外，我们还发现，随着 LDAO 浓度的增加，解折叠的协同性存在差异，这意味着胶束浓度和胶束-蛋白质比对桶的稳定性很重要。我们的结果表明，hVDAC-2 的九个半胱氨酸残基是与环境建立强（更强）桶相互作用的关键，并且还赋予桶支架额外的柔韧性。
