Veeraraghavan S, Rodriguez-Ghidarpour S, MacKinnon C, McGee W A, Pierce M M, Nall B T
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Biochemistry. 1995 Oct 3;34(39):12892-902. doi: 10.1021/bi00039a052.
Catalysis of slow folding reactions by peptidyl prolyl cis-trans isomerase (PPI) provides estimates of stabilities of intermediates in folding of normal and mutational variants of yeast iso-2 cytochrome c. A two-state model postulating a rapid preequilibration of intermediates with the unfolded protein is employed to calculate the stabilization free energy of the intermediate from the catalytic efficiency (kcat/Km) of PPI toward slow folding species. Stability measurements have been made for two distinct slow-folding intermediates: the absorbance-detected (IIS) and fluorescence-detected (IIIS) intermediates. Mutation-induced changes in the stability of the intermediates and in the activation free energy for slow folding are compared to changes in equilibrium thermodynamic stability. The results show that (1) for iso-2 the absorbance-detected intermediates (IIS) are slightly more stable than the fluorescence-detected intermediates (IIIS), (2) most mutations have different effects on equilibrium stability and the stability of the IIS or IIIS intermediates, and (3) for both slow folding reactions the mutation-induced changes in the activation free energy are small compared to the magnitude of the activation free energy barrier. Differential effects of mutations on equilibrium stability and the stability of intermediates provides a means of assessing the sequence-encoded structural specificity for folding. Mutations with different effects on intermediate stability and equilibrium stability change the encoded folding information and may alter folding pathways and/or lead to different three-dimensional structures. Identification of mutations which stabilize a folding intermediate relative to the native conformation provides an empirical approach to the design of thermodynamically stable forms of folding intermediates.
肽基脯氨酰顺反异构酶（PPI）对缓慢折叠反应的催化作用，为酵母同工酶-2细胞色素c正常变体和突变变体折叠过程中中间体的稳定性提供了估计。采用一种双态模型，该模型假定中间体与未折叠蛋白能快速达到预平衡，以根据PPI对缓慢折叠物种的催化效率（kcat/Km）来计算中间体的稳定化自由能。已对两种不同的缓慢折叠中间体进行了稳定性测量：吸光度检测到的（IIS）中间体和荧光检测到的（IIIS）中间体。将突变引起的中间体稳定性变化以及缓慢折叠的活化自由能变化与平衡热力学稳定性变化进行了比较。结果表明：（1）对于同工酶-2，吸光度检测到的中间体（IIS）比荧光检测到的中间体（IIIS）略稳定；（2）大多数突变对平衡稳定性以及IIS或IIIS中间体的稳定性有不同影响；（3）对于这两种缓慢折叠反应，与活化自由能垒的大小相比，突变引起的活化自由能变化较小。突变对平衡稳定性和中间体稳定性的不同影响提供了一种评估折叠的序列编码结构特异性的方法。对中间体稳定性和平衡稳定性有不同影响的突变会改变编码的折叠信息，并可能改变折叠途径和/或导致不同的三维结构。鉴定相对于天然构象稳定折叠中间体的突变，为设计热力学稳定形式的折叠中间体提供了一种经验方法。
