ACID-INDUCED UNFOLDING AND REFOLDING TRANSITIONS OF CYTOCHROME-C - A 3-STATE MECHANISM IN H2O AND D2O
ACID-INDUCED UNFOLDING AND REFOLDING TRANSITIONS OF CYTOCHROME-C - A 3-STATE MECHANISM IN H2O AND D2O
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DOI：
10.1021/bi00095a017
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发表时间：
1993-11-09
期刊：
影响因子：
2.9
通讯作者：
NISHII, I
中科院分区：
文献类型：
--
作者：
GOTO, Y;HAGIHARA, Y;NISHII, I
而酸变性的马铁细胞色素c在pH=2时的依赖于盐的构象转变是由酸未折叠状态到熔融球状状态的二态机制[Kataoka，M.，Hagihara，Y.，Mihara，K.，&Goto，Y.(1993)J.Mol.比奥尔。229,591-596]，D2O中相应的转变被认为涉及一个非致密的，α-螺旋中间态(预熔融球状态)[Jeng，M.-F.，&England ander，S.W.(1991)J.Mol.比奥尔。221、1045-1061]。为了检验构象转变中的差异，我们分别进行了H2O和D2O中细胞色素c的HCl和DCL滴定，通过远紫外圆二色谱、色氨酸荧光和Soret吸收进行了测量。在D2O和H2O中，观察到从天然状态到酸未折叠状态，然后再折叠到熔融球状状态。在任何一种溶剂中，构象转变都可以用最小1三态机制很好地近似，该三态机制由自然态、熔融态和酸展开态组成。因此，我们的结果没有证实D2O中存在预熔的球状状态。细胞色素c氨基的乙酰化在pH=2时可以稳定熔融的球状状态。根据三态机理，我们构建了pH和乙酰化度的构象相图。该相图与细胞色素c依赖于pH和盐的构象转变的相图相似，表明乙酰化对构象状态的影响类似于盐的影响。
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 minima.1 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.