Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit.
Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit.
复制标题
色氨酸合酶α亚基保守脯氨酸突变体的去折叠和重折叠的平衡和动力学分析。
DOI：
10.1021/bi961660c
复制
发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
K. Yutani
中科院分区：
文献类型：
--
作者：
K. Ogasahara;K. Yutani
为了阐明大肠杆菌色氨酸合成酶α亚基保守脯氨酸残基在稳定性和折叠中的作用，利用肽基圆二色性和芳族荧光测量在pH 7和25℃下对6个突变α亚基（Pro- >Ala）进行了盐酸胍诱导的展开-再折叠的平衡和动力学研究。(I)对于所有突变型和野生型蛋白，原生(N)和I态在水中的吉布斯能变化（δ Gni(H2O)）与展开（δ G++u(H2O)）和折叠（δ G++r(H2O)）反应在水中的激活吉布斯能变化（δ G++u(H2O)）的差值(δ G++u（H2O)- δ G++r(H2O)）一致。这意味着α亚基的早期折叠中间体对应于平衡中间体。与野生型相比，突变蛋白的δ Gni（H2O）值均降低。在I和变性(D)态之间，水的吉布斯能变化（δ Gid(H2O)）基本上不受取代的影响。δ G++u（H2O）和δ G++r（H2O）在所有突变蛋白中分别降低和增加。(2) 6种保守的脯氨酸以不同的方式参与α亚基的稳定性和折叠，其中脯氨酸28和96稳定α亚基的N态，脯氨酸28、96、132和207破坏α亚基的I态。脯氨酸57和62对稳定性的贡献很小。(3)顺式脯氨酸28不是由脯氨酸顺反异构化反应引起的重折叠动力学慢相的起源。
To elucidate the role of conserved proline residues of the tryptophan synthase alpha subunit from Escherichia coli in stability and folding, equilibrium and kinetic studies of the unfolding-refolding induced by guanidine hydrochloride for six mutant alpha subunits (Pro-->Ala) were carried out by peptidyl circular dichroism and aromatic fluorescence measurements at pH 7 and 25 degrees C. These results were analyzed assuming the presence of one intermediate (I) state in the denaturation process. (I) For all mutant and wild-type proteins, the Gibbs energy change (delta Gni(H2O)) in water between the native (N) and I states coincided with the difference (delta G++u(H2O)-delta G++r(H2O)) between the activation Gibbs energy changes in water for the unfolding (delta G++u(H2O) and refolding (delta G++r(H2O) reactions. This means that the early folding intermediate of the alpha subunit corresponds to the equilibrium intermediate. Delta Gni(H2O) values of all mutant proteins decreased compared with that of the wild-type protein. Gibbs energy change (delta Gid(H2O) in water between I and the denatured (D) states was not substantially affected by the substitutions. Delta G++u(H2O) and delta G++r(H2O) decreased and increased, respectively, for all mutant proteins. (2) Six conserved prolines played roles in stability and folding of the alpha subunit in a different manner: prolines 28 and 96 by stabilizing the N state and prolines 28, 96, 132, and 207 by destabilizing the I state. The contributions of prolines 57 and 62 to the stability were marginal. (3) Cis proline 28 was not the origin of the slow phase in the refolding kinetics assumed to arise from the cis-trans isomerization reaction of proline.