The search for local native-like nucleation centers in the unfolded state of β-sheet proteins

An approach involving the systematic computational conformational analysis of all overlapping hexapeptide segments in the protein sequence has found fragments with the higher than average propensity to adopt the native-like three-dimensional structure and other regular nonrandom structures in the unfolded states of four β-sheet proteins, namely IFABP (intestinal fatty acid-binding protein), ILBP (ileal fatty acid-binding protein), CRABP I (cellular retinoic acid-binding protein), and CRBP II (cellular retinal binding protein). The native three-dimensional structures of these four proteins are very similar even though they possess as little as ≈30% sequence similarity. The computational results were validated by comparison with the experimental data of the heteronuclear sequential quantum correlation NMR spectroscopy obtained earlier for IFABP at high urea concentrations. On this basis, a molecular model of the unfolded state of IFABP has been developed. The model presumes a dynamic equilibrium between various nonrandom structures (including the native-like structure) and random coil in the local segments of the protein sequence. The model explains experimental observations obtained earlier for folding of several mutants of IFABP, as well as the observed differences in molecular mechanisms of folding for the four β-sheet proteins. Because the computational approach itself does not employ any experimentally derived information in advance, it is not necessarily limited to the β-sheet proteins.

本研究采用针对蛋白质序列中所有重叠六肽区段（hexapeptide segments）开展系统性计算构象分析的方法，在四种β折叠蛋白（β-sheet proteins）的解折叠态中，筛选出了具有高于平均形成倾向的、可形成类天然三维结构及其他规则非随机结构的肽段。本次研究涉及的四种β折叠蛋白分别为IFABP（肠脂肪酸结合蛋白，intestinal fatty acid-binding protein）、ILBP（回肠脂肪酸结合蛋白，ileal fatty acid-binding protein）、CRABP I（细胞视黄酸结合蛋白I，cellular retinoic acid-binding protein）以及CRBP II（细胞视黄醛结合蛋白，cellular retinal binding protein）。尽管这四种蛋白的序列相似度仅约30%，但它们的天然三维结构却高度相似。本研究通过将计算结果与此前在高尿素浓度条件下针对IFABP测得的异核顺序量子相关核磁共振波谱（heteronuclear sequential quantum correlation NMR spectroscopy）实验数据进行对比，验证了计算结果的可靠性。在此基础上，我们构建了IFABP解折叠态的分子模型。该模型假设蛋白质序列的局部区段中，各类非随机结构（包含类天然结构）与无规卷曲之间处于动态平衡。该模型能够解释此前针对IFABP多种突变体折叠过程得到的实验观测结果，同时也可阐释这四种β折叠蛋白折叠分子机制的差异。由于该计算方法本身未预先引入任何实验衍生信息，因此其应用范围未必局限于β折叠蛋白。