Evaluation of cooperative interactions between substructures of iso-1-cytochrome c using double mutant cycles.
Evaluation of cooperative interactions between substructures of iso-1-cytochrome c using double mutant cycles.
复制标题
使用双突变体循环评估 iso-1-细胞色素 c 亚结构之间的协同相互作用。
DOI：
10.1021/bi034958t
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Bowler,BruceE
中科院分区：
文献类型：
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作者：
Wandschneider,Eydiejo;Hammack,BarbaraN;Bowler,BruceE
双突变周期被用来评估全局稳定突变天门汀52→异亮氨酸（N52I）与在26、33、39、54、73、89和100位产生单一表面组氨酸的突变之间的相互作用能。这些组氨酸突变位点分布在细胞染色质的四个协同折叠单元上。双突变周期从异-1细胞色素变异体AcTM开始，该变异体不含表面组氨酸，在52号位置含有天冬酰胺。异亮氨酸单独添加在52号位置，AcTMI52变体，表面组氨酸，AcHX变体，其中X表示组氨酸序列位置。双突变变体，AcHXI52，提供了双突变循环的剩余角落。用盐酸胍变性法测定各变异的稳定性，并计算52位与各组氨酸位点的相互作用能。7个双突变体中有6个表现出组氨酸突变与N52I突变的可加性稳定性效应（AcH33I52、AcH39I52、AcH54I52、AcH89I52和AcH100I52）或弱互作能（AcH73I52），这与蛋白质折叠的协同效应和稳定性在蛋白质结构上的稀疏分布一致。AcH26I52突变体在一个亚结构上的N52I突变与相邻亚结构上的His 26的添加之间表现出很强的有利相互作用能，为2.0±0.5 kcal/mol。这些数据与il52突变导致His 26和Glu 44之间亚结构间氢键的熵稳定一致。
A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 → isoleucine (N52I) in iso-1-cytochromecand mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100. These histidine mutation sites are distributed through the four cooperative folding units of cytochromec. The double mutant cycle starts with the iso-1-cytochromecvariant AcTM, a variant with no surface histidines and with asparagine at position 52. Isoleucine is added singly at position 52, AcTMI52 variant, as are the surface histidines, AcHX variants, where X indicates the histidine sequence position. The double mutant variants, AcHXI52, provide the remaining corner of the double mutant cycle. The stabilities of all variants were determined by guanidine hydrochloride denaturation and interaction energies were calculated between position 52 and each histidine site. Six of the seven double mutants show additive (AcH33I52, AcH39I52, AcH54I52, AcH89I52, and AcH100I52) stability effects or weak interaction energies (AcH73I52) of the histidine mutations and the N52I mutation, consistent with cooperative effects on protein folding and stability being sparsely distributed through the protein structure. The AcH26I52 variant shows a strong favorable interaction energy, 2.0 ± 0.5 kcal/mol, between the N52I mutation in one substructure and the addition of His 26 to an adjacent substructure. The data are consistent with an entropic stabilization of the intersubstructure hydrogen bond between His 26 and Glu 44 by the Ile 52 mutation.