Farrer Brian T, Pecoraro Vincent L
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3760-5. doi: 10.1073/pnas.0336055100. Epub 2003 Jan 27.
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 CH(3)CO-G LKALEEK CKALEEK LKALEEK G-NH(2) (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 (deltaH = 20(2) kcalmol; deltaS= 40(5) calmol K) and the rearrangement of the misfolded intermediate steps (deltaH = 23(2) kcalmol; deltaS= 27(5) calmol K). The mechanism is discussed with regard to metalloprotein folding and metalloprotein design.
本文描述了对一种从头设计的蛋白质进行金属包封的详细动力学分析。通过全局分析推导了肽CH(3)CO-G LKALEEK CKALEEK LKALEEK G-NH(2)（小L9C）形成的三链卷曲螺旋中Hg(II)包封的动力学机制。该机制涉及两个肽段被Hg(II)迅速初始折叠，形成Hg(Baby L9C(-H))(2)，其中线性硫醇汞(II)与半胱氨酸硫原子结合。这里，Baby L9C(-H)表示半胱氨酸硫醇去质子化的Baby L9C。添加第三个肽段形成三链卷曲螺旋是速率决定步骤，并产生一个涉及两个独立物种的中间状态。其中一个物种，称为正确折叠的中间体，第三个半胱氨酸硫醇迅速去质子化，产生所需的带有包封三角硫醇汞(II)的三链卷曲螺旋。另一个物种，称为错误折叠的中间体，在一个实验上可区分的步骤中重排为正确折叠的中间体。涉及添加第三个肽段的反应相对于Baby L9C浓度的反应级数表明，第三个螺旋的添加仅通过Hg(Baby L9C(-H))(2)与未与卷曲螺旋结合的Baby L9C反应发生。反应的温度依赖性给出了添加第三个螺旋（ΔH = 20(2) kcal/mol；ΔS = 40(5) cal/mol K）和错误折叠中间体步骤重排（ΔH = 23(2) kcal/mol；ΔS = 27(5) cal/mol K）的活化参数。本文还讨论了该机制与金属蛋白折叠和金属蛋白设计的关系。
