Mutational analysis of the BPTI folding pathway .2. Effects of aromatic->leucine substitutions on folding kinetics and thermodynamics

被引:17
作者
Zhang, JX [1 ]
Goldenberg, DP [1 ]
机构
[1] UNIV UTAH,DEPT BIOL,SALT LAKE CITY,UT 84112
关键词
aromatic residues; bovine pancreatic trypsin inhibitor; disulfide bonds; folding kinetics; hydrophobic residues; protein folding; stability;
D O I
10.1002/pro.5560060720
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The rates of the individual steps in the disulfide-coupled folding and unfolding of eight BPTI variants, each containing a single aromatic to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide-bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they Bad significantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically-preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild-type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.
引用
收藏
页码:1563 / 1576
页数:14
相关论文
共 57 条