Ramirez-Alvarado M, Daragan V A, Serrano L, Mayo K H
Department of Biochemistry, Biomedical Engineering Center, University of Minnesota, Minneapolis 55455, USA.
Protein Sci. 1998 Mar;7(3):720-9. doi: 10.1002/pro.5560070321.
Structurally characterizing partially folded peptides is problematic given the nature of their transient conformational states. 13C-NMR relaxation data can provide information on the geometry of bond rotations, motional restrictions, and correlated bond rotations of the backbone and side chains and, therefore, is one approach that is useful to assess the presence of folded structure within a conformational ensemble. A peptide 12mer, R1GITVNG7KTYGR12, has been shown to partially fold in a relatively stable beta-hairpin conformation centered at NG. Here, five residues, G2, V5, G7, Y10, G11, were selectively 13C-enriched, and 13C-NMR relaxation experiments were performed to obtain auto- and cross-correlation motional order parameters, correlation times, bond rotation angular variances, and bond rotational correlation coefficients. Our results indicate that, of the three glycines, G7 within the hairpin beta-turn displays the most correlated phi(t),psi(t) rotations with its axis of rotation bisecting the angle defined by the H-C-H bonds. These positively correlated bond rotations give rise to "twisting" type motions of the HCH group. V5 and Y10 phi,psi bond rotations are also positively correlated, with their CbetaCalphaH groups undergoing similar "twisting" type motions. Motions of near-terminal residues G2 and G11 are less restricted and less correlated and are best described as wobbling-in-a-cone. V5 and Y10 side-chain motions, aside from being highly restricted, were found to be correlated with phi,psi bond rotations. At 303 K, where the hairpin is considered "unfolded," the peptide exists in a transient, collapsed state because backbone and side-chain motions of V5, G7, and Y10 remain relatively restricted, unlike their counterparts in GXG-based tripeptides. These results provide unique information toward understanding conformational variability in the unfolded state of proteins, which is necessary to solve the protein folding problem.
鉴于部分折叠肽的瞬态构象状态的性质，对其进行结构表征存在问题。13C-NMR弛豫数据可以提供有关键旋转的几何结构、运动限制以及主链和侧链的相关键旋转的信息，因此，这是一种用于评估构象集合中折叠结构存在情况的有用方法。一种12聚体肽R1GITVNG7KTYGR12已被证明以围绕NG的相对稳定的β-发夹构象部分折叠。在这里，五个残基G2、V5、G7、Y10、G11被选择性地进行13C富集，并进行13C-NMR弛豫实验以获得自相关和交叉相关的运动序参数、相关时间、键旋转角方差和键旋转相关系数。我们的结果表明，在三个甘氨酸中，发夹β-转角内的G7显示出最相关的φ(t)、ψ(t)旋转，其旋转轴平分由H-C-H键定义的角度。这些正相关的键旋转导致HCH基团的“扭曲”型运动。V5和Y10的φ、ψ键旋转也呈正相关，它们的CβCaαH基团经历类似的“扭曲”型运动。近末端残基G2和G11的运动限制较少且相关性较低，最好描述为圆锥内摆动。除了受到高度限制外，还发现V5和Y10的侧链运动与φ、ψ键旋转相关。在303 K时，发夹被认为是“未折叠的”，该肽以瞬态、塌缩状态存在，因为V5、G7和Y10的主链和侧链运动仍然相对受限，这与基于GXG的三肽中的对应部分不同。这些结果为理解蛋白质未折叠状态下的构象变异性提供了独特的信息，这对于解决蛋白质折叠问题是必要的。
