Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c.
Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c.
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天然或类天然物种是碱性 iso-2 细胞色素 c 折叠中的瞬时中间体。
DOI：
10.1021/bi00358a036
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Nall,BT
中科院分区：
文献类型：
--
作者：
Nall,BT
1986年1月14日收到的修订手稿摘要：在高pH下滴定将酵母iso-2细胞色素c转化为缺乏695 nm吸收带的不活跃但更稳定的碱性形式[Osterhout，JJ，Jr.，Muthukrishnan，K.，&Nall，B.T.(1985)生化24,6680-6684]，通过在停流仪中稀释盐酸胍，测量了碱性形式的吸光度检测复性的动力学。检测到快速折叠物种(R2)，就像在中性pH下重折叠到本土化一样。观察到一个附加的动力学相(Ra)，其幅度与快相的符号相反。该相的幅值随pH的升高而增大，速率随pH的升高而增大。对完全折叠的蛋白质的pH跳跃测量表明，Ra相与碱性异构化反应具有相同的符号、速率和pH依赖性，表明这一新相涉及快速折叠后的天然或类天然物种的异构化。在高pH条件下的复性过程中，以5-S间隔拍摄了吸光度差光谱。光谱证实，在蛋白质转化为碱性形式之前，具有695 nm吸收带的类本地化物种是瞬时形成的。在抗坏血酸存在下的重折叠表明，与碱性iso-2不同，瞬时的、类本地的物种是可还原的。因此，(1)再折叠成碱性形式的iso-2细胞色素c是通过短暂的天然或类天然物种进行的，以及(2)导致天然或类天然形式的折叠途径在高pH下保持不变，其中天然物种不再是热力学上的有利产物。对于折叠的后期阶段，结果反对仅在热力学支持的产品中发现的结构的增长和合并组装[Harison，S.C.，&Durbin，R.(1985)Proc。娜塔莉。阿卡德。SCI。美国82,4028-4030]，并表明存在独特的物种，它们引导折叠--可能沿着一条连续的路径[Kim，PS，&Baldwin，R.L.(1982)Annu。Biochem牧师。51,459-489]，
Revised Manuscript Received January 14, 1986 abstract: Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, JJ, Jr., Muthukrishnan, K., &Nall, B. T.(1985) Biochemistry 24, 6680-6684], The kinetics of absorbance-detected refolding of the alkalineform have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (r2) are detected, as in refolding to the nativestate at neutralpH. An additional kinetic phase (ra) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparisonto pH-jump measurements of the fully folded protein shows that phase ra has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species—with a 695-nm absorbance band—are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus,(1) refolding to the alkaline form of iso-2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leadingto native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product. For the latter stages of folding, the results argue against growth and merge assembly of structure found only in the thermodynamically favored product [Harrison, S. C., & Durbin, R.(1985) Proc. Natl. Acad. Sci. USA 82, 4028-4030] andsuggest the existence of unique species which direct folding—perhaps along a sequential pathway [Kim, PS, & Baldwin, R. L.(1982) Annu. Rev. Biochem. 51, 459-489],