Cytochrome c folding traps are not due solely to histidine-heme ligation: direct demonstration of a role for N-terminal amino group-heme ligation.
Cytochrome c folding traps are not due solely to histidine-heme ligation: direct demonstration of a role for N-terminal amino group-heme ligation.
复制标题
细胞色素 c 折叠陷阱不仅仅归因于组氨酸-血红素连接：直接证明了 N 末端氨基-血红素连接的作用。
DOI：
10.1006/jmbi.1997.1493
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Bowler,BE
中科院分区：
文献类型：
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作者：
Hammack,B;Godbole,S;Bowler,BE
在以前的工作中，血红素连接对细胞色素c折叠的影响已被归因于组氨酸侧链。酵母异-1-细胞色素c的一种变体TM，除了His 18外，它缺乏所有组氨酸残基，在pH 5 - 6之间的范围内仍然显示出变性状态血红素连接的证据，其中通常只预期组氨酸连接。TM的N-末端氨基通过与乙醛酸酯的转氨反应转化为羰基，产生不具有末端氨基的蛋白质（ModTM）。由于这种修饰，在3 M胍-HCl中血红素连接损失的中点pH（pH 1/2）从5.9变为7.4，为在这些条件下N-末端氨基-血红素连接提供了直接证据。因此，在变性条件下，N-末端氨基与组氨酸竞争异-1-细胞色素c的错连。为了评估变性状态N-末端氨基-血红素连接对异-1-细胞色素c折叠的影响，进行了停流动力学实验。在pH6.2时，ModTM、TM和野生型蛋白的主要复性寿命（3 M → 0.27M盐酸胍）分别为11.6ms、30 ms和1.3s。因此，N-末端氨基的变性状态连接使折叠减慢2.6倍。
In previous work, heme ligation effects on the folding of cytochrome c have been attributed to histidine side-chains. A variant of yeast iso-1-cytochrome c designated TM, which lacks all histidine residues except His18, still shows evidence of denatured state heme ligation in the pH range between 5 and 6 where normally only histidine ligation is expected. Conversion of the N-terminal amino group of TM to a carbonyl group through a transamination reaction with glyoxylate produced a protein (ModTM) with no terminal amino group. The midpoint pH (pH1/2) for loss of heme ligation in 3 M guanidine-HCl shifts from 5.9 to 7.4 as a result of this modification, providing direct evidence for N-terminal amino group-heme ligation under these conditions. The N-terminal amino group thus competes with histidine for misligation of iso-1-cytochrome c under denaturing conditions. To assess the effect of denatured state N-terminal amino group-heme ligation on the folding of iso-1-cytochrome c, stopped-flow kinetics experiments were conducted. At pH 6.2, the major refolding lifetimes (3 M → 0.27 M guanidine-HCl) for ModTM, TM and the wild-type protein are 11.6 ms, 30 ms and 1.3 seconds, respectively. Denatured state ligation of the N-terminal amino group thus slows folding 2.6-fold.