Hodel A, Rice L M, Simonson T, Fox R O, Brünger A T
Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA.
Protein Sci. 1995 Apr;4(4):636-54. doi: 10.1002/pro.5560040405.
Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation.
葡萄球菌核酸酶A以两种折叠形式存在，这两种形式在赖氨酸116 - 脯氨酸117肽键的异构化状态上有所不同。占主导地位的形式（占有率90%）采用顺式肽键，这在晶体结构中可以观察到。核磁共振研究表明，顺式和反式形式之间的自由能差异相对较小（ΔGcis→trans约为1.2千卡/摩尔），这是由焓和熵的巨大且近乎补偿性的差异导致的（ΔHcis→trans约为ΔTScis→trans约为10千卡/摩尔）。X射线晶体结构的证据表明，核酸酶顺式和反式形式之间的结构差异仅限于112 - 117位残基的构象，这是一个溶剂化的蛋白质环。在这里，我们通过详尽的构象搜索获得了这个蛋白质环构象平衡的热力学和结构描述，该搜索确定了几个亚状态，随后对这些亚状态之间进行了自由能模拟。通过将搜索划分为构象亚状态，我们在这种特殊情况下克服了多个极小值问题，并获得了精确且可重复的自由能值。通过在明确处理的环与蛋白质其余部分之间适当选择非键合项，对蛋白质和水环境进行了隐式建模。这些模拟正确地预测了顺式和反式形式之间的小自由能差异，该差异由焓和熵的较大且补偿性的差异组成。这些模拟的结构预测在定性上与核酸酶变体已知的X射线结构一致，并产生了未知的次要反式构象的模型。
