Goto Y, Hagihara Y, Hamada D, Hoshino M, Nishii I
Department of Biology, Faculty of Science, Osaka University, Japan.
Biochemistry. 1993 Nov 9;32(44):11878-85. doi: 10.1021/bi00095a017.
Whereas the salt-dependent conformational transition of acid-denatured horse ferricytochrome c at pH 2 is approximated by a two-state mechanism from the acid-unfolded state to the molten globule state [Kataoka, M., Hagihara, Y., Mihara, K., & Goto, Y. (1993) J. Mol. Biol. 229, 591-596], the corresponding transition in D2O has been proposed to involve a noncompact, alpha-helical intermediate state (the pre-molten globule state) [Jeng, M.-F., & Englander, S. W. (1991) J. Mol. Biol. 221, 1045-1061]. To examine the proposed difference in the conformational transitions, we carried out the HCl and DCl titrations of cytochrome c in H2O and D2O, respectively, measured by far-UV circular dichroism, tryptophan fluorescence, and Soret absorption. In both D2O and H2O, unfolding from the native state to the acid-unfolded state and subsequent refolding to the molten globule state were observed. In either solvent, the conformational transitions were well approximated by a minimal three-state mechanism consisting of the native, molten globule, and acid-unfolded states. Thus, our results did not substantiate the presence of a pre-molten globule state in D2O. Acetylation of amino groups of cytochrome c is known to stabilize the molten globule state at pH 2. On the basis of the three-state mechanism, we constructed a conformational phase diagram for the effect of pH and the degree of acetylation. This phase diagram was similar to that of the pH- and salt-dependent conformational transition of cytochrome c, suggesting that the effects of acetylation on the conformational states are similar to those of salt.
在pH 2时，酸变性的马铁细胞色素c的盐依赖性构象转变可通过从酸解折叠状态到熔球状态的两态机制近似描述[片冈，M.，萩原，Y.，三原，K.，& 后藤，Y.（1993）《分子生物学杂志》229，591 - 596]，而在D2O中的相应转变被认为涉及一个非紧密的α螺旋中间态（预熔球状态）[郑，M.-F.，& 英格兰德，S. W.（1991）《分子生物学杂志》221，1045 - 1061]。为了研究构象转变中所提出的差异，我们分别在H2O和D2O中对细胞色素c进行了HCl和DCl滴定，并通过远紫外圆二色性、色氨酸荧光和Soret吸收进行测量。在D2O和H2O中，均观察到从天然状态解折叠至酸解折叠状态以及随后再折叠至熔球状态的过程。在任何一种溶剂中，构象转变都能很好地通过由天然态、熔球态和酸解折叠态组成的最小三态机制近似描述。因此，我们的结果并未证实D2O中存在预熔球状态。已知细胞色素c的氨基乙酰化可在pH 2时稳定熔球状态。基于三态机制，我们构建了一个关于pH和乙酰化程度影响的构象相图。该相图与细胞色素c的pH和盐依赖性构象转变的相图相似，表明乙酰化对构象状态的影响与盐的影响相似。
