Boice J A, Dieckmann G R, DeGrado W F, Fairman R
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Biochemistry. 1996 Nov 19;35(46):14480-5. doi: 10.1021/bi961831d.
The study and successful design of coiled-coil protein structural motifs have provided much insight into the rules governing protein folding and stability. In this work we use a thermodynamic approach to quantitate the rules that govern the specific oligomerization of coiled coils. We have designed a highly stable trimeric coiled coil by placing valine residues at each a position and leucine residues at each d position of the heptad repeating unit. The peptide forms a very stable trimer as determined by sedimentation equilibrium, and the concentration dependence of its circular dichroism spectrum follows a cooperative monomer/dimer/trimer equilibrium with the dimer state as a highly unstable intermediate. Its guanidinium chloride denaturation curve was collected at several peptide concentrations, and analysis of the data confirms the cooperativity of the trimerization process and provides a free energy of stabilization of - 18.4 kcal mol-1 for the trimer. The heat capacity, delta Cp, was measured by global analysis of thermal unfolding data collected at a number of guanidinium chloride concentrations. Guanidinium chloride induces cold denaturation in the thermal unfolding curves, providing a reasonably well-determined value for delta Cp of 750 cal deg-1 mol-1. This translates to a delta Cp of 8.6 cal deg-1 mol-1 per residue and corresponds well to that expected of a coiled coil with a well-defined tertiary structure.
对卷曲螺旋蛋白结构基序的研究和成功设计，为了解蛋白质折叠和稳定性的规律提供了很多见解。在这项工作中，我们采用热力学方法来量化控制卷曲螺旋特定寡聚化的规律。我们通过在七肽重复单元的每个a位放置缬氨酸残基，在每个d位放置亮氨酸残基，设计了一种高度稳定的三聚体卷曲螺旋。通过沉降平衡测定，该肽形成了非常稳定的三聚体，其圆二色光谱的浓度依赖性遵循协同的单体/二聚体/三聚体平衡，其中二聚体状态是高度不稳定的中间体。在几种肽浓度下收集了其氯化胍变性曲线，对数据的分析证实了三聚化过程的协同性，并为三聚体提供了-18.4千卡/摩尔的稳定自由能。通过对在多个氯化胍浓度下收集的热变性数据进行全局分析，测量了热容ΔCp。氯化胍在热变性曲线中诱导冷变性，为ΔCp提供了一个合理确定的值，即750卡/开尔文/摩尔。这相当于每个残基的ΔCp为8.6卡/开尔文/摩尔，与具有明确三级结构的卷曲螺旋预期值相符。
