期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2003
卷号：100
期号：7
页码：3760-3765
DOI：10.1073/pnas.0336055100
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：A detailed kinetic analysis of metal encapsulation by a de novo-designed protein is described. The kinetic mechanism of Hg(II) encapsulation in the three-stranded coiled coil formed by the peptide CH3CO-G LKALEEK CKALEEK LKALEEK G-NH2 (Baby L9C) is derived by global analysis. The mechanism involves rapid initial collapse of two peptides by Hg(II) forming Hg(Baby L9C-H)2 with a linear thiolato Hg(II) bound to the cysteine sulfur atoms. Here, Baby L9C-H denotes Baby L9C with the cysteine thiol deprotonated. Addition of the third peptide, forming the three-stranded coiled coil, is the rate-determining step and results in an intermediate state involving two separate species. One of the species, termed the properly folded intermediate, undergoes rapid deprotonation of the third cysteine thiol, yielding the desired three-stranded coiled coil with an encapsulated trigonal thiolato Hg(II). The other species, termed the misfolded intermediate, rearranges in an experimentally distinguishable step to the properly folded intermediate. The order of the reaction involving the addition of the third peptide with respect to the concentration of Baby L9C indicates that addition of the third helix only occurs through reaction of Hg(Baby L9C-H)2 and Baby L9C that is unassociated with a coiled coil. Temperature dependence of the reaction afforded activation parameters for both the addition of the third helix ({Delta}H{ddagger} = 20(2) kcal/mol; {Delta}S{ddagger} = 40(5) cal/mol K) and the rearrangement of the misfolded intermediate steps ({Delta}H{ddagger} = 23(2) kcal/mol; {Delta}S{ddagger} = 27(5) cal/mol K). The mechanism is discussed with regard to metalloprotein folding and metalloprotein design.
