Wong K B, Freund S M, Fersht A R
MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, UK.
J Mol Biol. 1996 Jun 21;259(4):805-18. doi: 10.1006/jmbi.1996.0359.
Detection of residual structure in denatured proteins is of interest because fleetingly structured regions may be initiation points of the folding pathway. Residual structure in this context is not the definition of one stable conformation but a population phenomenon. Acid, thermal and solvent-denatured states have recently been examined by NMR spectroscopy, but cold-denatured states have not been characterised to date. Cold denaturation is a general phenomenon of globular proteins, which provides a convenient route for studying early events in protein folding: such states can be induced to fold and be monitored on a submillisecond time scale by temperature-jump methods. Here, we use NMR spectroscopy to define residual structure in cold-denatured barstar. The cold-denatured state becomes significantly populated in the presence of increasing concentrations of urea and lower temperature. In the presence of 3 M urea, the double mutant of barstar in which Cys40 and Cys82 are both mutated to Ala (C40/82A) is completely and reversibly denatured at 278 K, a temperature that is accessible to NMR experiments. This cold-denatured state of barstar was assigned by heteronuclear NMR experiments and structural parameters such as NOE, coupling constants and chemical shifts were derived. Based on the excellent dispersion in a HSQC-NOESY-HSQC experiment, dNN(i,i+1) NOEs were observed for almost all residues. This allowed us to normalise NOE intensities as the NOE: diagonal ratio dNN(i,i+1) NOE (sigma NN) and the NOE ratio of d(alpha N(i+1,i+1)):d(alpha N(i,i+1)) (sigma N alpha/sigma alpha N). This approach reveals residual structure populating the alpha-region of the (phi, psi) conformational space in the regions corresponding to the first and the second helices and near the end of the second beta-strand of native barstar, whereas the C-terminal region that corresponds to the fourth helix and the third beta-strand is in a random coil conformation. The results suggest that the first and the second helices are potential initiation sites for the folding of barstar. The details presented here provide the starting point for the study of rapid folding of cold-denatured barstar.
检测变性蛋白质中的残余结构备受关注，因为瞬间形成的结构化区域可能是折叠途径的起始点。在此背景下，残余结构并非指一种稳定构象的定义，而是一种群体现象。酸、热和溶剂变性状态最近已通过核磁共振光谱法进行研究，但冷变性状态迄今尚未得到表征。冷变性是球状蛋白质的普遍现象，它为研究蛋白质折叠早期事件提供了一条便捷途径：通过温度跳跃方法，可以诱导这些状态折叠并在亚毫秒时间尺度上进行监测。在此，我们使用核磁共振光谱法来确定冷变性巴氏杆菌核糖核酸酶抑制剂（barstar）中的残余结构。在尿素浓度增加和温度降低的情况下，冷变性状态的数量显著增加。在存在3 M尿素的情况下，巴氏杆菌核糖核酸酶抑制剂中Cys40和Cys82均突变为Ala的双突变体（C40/82A）在278 K时完全且可逆地变性，这一温度是核磁共振实验可达到的。通过异核核磁共振实验确定了巴氏杆菌核糖核酸酶抑制剂的这种冷变性状态，并得出了诸如核Overhauser效应（NOE）、耦合常数和化学位移等结构参数。基于HSQC-NOESY-HSQC实验中的出色分散性，几乎所有残基都观察到了dNN(i,i + 1) NOE。这使我们能够将NOE强度归一化为NOE:对角线比率dNN(i,i + 1) NOE（σNN）以及d(αN(i + 1,i + 1)):d(αN(i,i + 1))的NOE比率（σNα/σαN）。这种方法揭示了在天然巴氏杆菌核糖核酸酶抑制剂的第一和第二螺旋区域以及第二β链末端附近，在(φ, ψ)构象空间的α区域中存在残余结构，而对应于第四螺旋和第三β链的C末端区域则处于无规卷曲构象。结果表明，第一和第二螺旋是巴氏杆菌核糖核酸酶抑制剂折叠的潜在起始位点。此处呈现的细节为研究冷变性巴氏杆菌核糖核酸酶抑制剂的快速折叠提供了起点。
