National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
Biochemistry. 2011 Apr 5;50(13):2434-44. doi: 10.1021/bi101955f. Epub 2011 Mar 10.
To improve our understanding of the contributions of different stabilizing interactions to protein stability, including that of residual structure in the unfolded state, the small sweet protein monellin has been studied in both its two variant forms, the naturally occurring double-chain variant (dcMN) and the artificially created single-chain variant (scMN). Equilibrium guanidine hydrochloride-induced unfolding studies at pH 7 show that the standard free energy of unfolding, ΔG°(U), of dcMN to unfolded chains A and B and its dependence on guanidine hydrochloride (GdnHCl) concentration are both independent of protein concentration, while the midpoint of unfolding has an exponential dependence on protein concentration. Hence, the unfolding of dcMN like that of scMN can be described as two-state unfolding. The free energy of dissociation, ΔG°(d), of the two free chains, A and B, from dcMN, as measured by equilibrium binding studies, is significantly lower than ΔG°(U), apparently because of the presence of residual structure in free chain B. The value of ΔG°(U), at the standard concentration of 1 M, is found to be ∼5.5 kcal mol(-1) higher for dcMN than for scMN in the range from pH 4 to 9, over which unfolding appears to be two-state. Hence, dcMN appears to be more stable than scMN. It seems that unfolded scMN is stabilized by residual structure that is absent in unfolded dcMN and/or that native scMN is destabilized by strain that is relieved in native dcMN. The value of ΔG°(U) for both protein variants decreases with an increase in pH from 4 to 9, apparently because of the thermodynamic coupling of unfolding to the protonation of a buried carboxylate side chain whose pK(a) shifts from 4.5 in the unfolded state to 9 in the native state. Finally, it is shown that although the thermodynamic stabilities of dcMN and scMN are very different, their kinetic stabilities with respect to unfolding in GdnHCl are very similar.
为了提高我们对不同稳定相互作用对蛋白质稳定性的贡献的理解，包括未折叠状态下残留结构的贡献，已经对小甜蛋白莫内林的两种变体形式，即天然存在的双链变体 (dcMN) 和人工创造的单链变体 (scMN) 进行了研究。在 pH 值为 7 时，用平衡盐酸胍诱导的展开研究表明，dcMN 到展开的链 A 和 B 的标准自由能展开，ΔG°(U)，以及它对盐酸胍 (GdnHCl) 浓度的依赖性都与蛋白质浓度无关，而展开的中点则与蛋白质浓度呈指数关系。因此，dcMN 的展开与 scMN 的展开一样，可以描述为两态展开。通过平衡结合研究测量的，dcMN 中两条游离链 A 和 B 的游离解离自由能，ΔG°(d)，明显低于 ΔG°(U)，显然是因为游离链 B 中存在残留结构。在标准浓度为 1 M 的情况下，在 pH 值为 4 到 9 的范围内，发现 dcMN 的 ΔG°(U)值比 scMN 高约 5.5 kcal mol(-1)，在这个范围内展开似乎是两态的。因此，dcMN 似乎比 scMN 更稳定。似乎展开的 scMN 由在展开的 dcMN 中不存在的残留结构稳定，或者天然的 scMN 由在天然的 dcMN 中缓解的应变不稳定。两种蛋白质变体的 ΔG°(U)值随 pH 值从 4 增加到 9 而降低，显然是由于展开与一个埋藏的羧酸盐侧链质子化的热力学偶联所致，其 pK(a)从展开状态的 4.5 转移到天然状态的 9。最后，结果表明，尽管 dcMN 和 scMN 的热力学稳定性非常不同，但它们在盐酸胍中展开的动力学稳定性非常相似。
