Baxter N J, Hosszu L L, Waltho J P, Williamson M P
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Sheffield, Western Bank, S10 2TN, UK.
J Mol Biol. 1998 Dec 18;284(5):1625-39. doi: 10.1006/jmbi.1998.2265.
It is demonstrated that the identity of residues accessing excited conformational states that are of low free energy relative to the ground state in proteins can be obtained from amide proton NMR chemical shift temperature dependences displaying significant curvature. For the N-terminal domain of phosphoglycerate kinase, hen egg-white lysozyme and BPTI, conformational heterogeneity arises from a number of independent sources, including: structural instability resulting from deletion of part of the protein; a minor conformer generated through disulphide bond isomerisation; an alternative hydrogen bond network associated with buried water molecules; alternative hydrogen bonds involving backbone amides and surface-exposed side-chain hydrogen bond acceptors; and the disruption of loops, ends of secondary structural elements and chain termini. In many of these cases, the conformational heterogeneity at these sites has previously been identified by X-ray and/or NMR studies, but conformational heterogeneity of buried water molecules has hitherto received little attention. These multiple independent low free-energy excited states each involve a small number of residues and are shown to be within 2.5 kcal mol-1 of the ground state. Their relationship with the partially unfolded forms previously characterised using amide proton exchange studies is discussed.
已证明，可从显示出显著曲率的酰胺质子核磁共振化学位移温度依赖性中获得蛋白质中相对于基态具有低自由能的激发构象状态下可及残基的身份。对于磷酸甘油酸激酶的N端结构域、鸡蛋清溶菌酶和抑肽酶，构象异质性源于多个独立来源，包括：蛋白质部分缺失导致的结构不稳定性；通过二硫键异构化产生的次要构象体；与埋藏水分子相关的替代氢键网络；涉及主链酰胺和表面暴露侧链氢键受体的替代氢键；以及环、二级结构元件末端和链端的破坏。在许多这些情况下，这些位点的构象异质性先前已通过X射线和/或核磁共振研究确定，但埋藏水分子的构象异质性迄今很少受到关注。这些多个独立的低自由能激发态各自涉及少数残基，并显示出与基态相差在2.5千卡摩尔-1以内。讨论了它们与先前使用酰胺质子交换研究表征的部分未折叠形式的关系。
