Mohana-Borges Ronaldo, Goto Natalie K, Kroon Gerard J A, Dyson H Jane, Wright Peter E
Department of Molecular Biology and Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 2004 Jul 23;340(5):1131-42. doi: 10.1016/j.jmb.2004.05.022.
The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between (15)N and (1)H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with phi and psi dihedral angles favoring the beta or P(II) regions. Each statistical segment has a highly anisotropic shape, with the N-H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the acid-denatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.
通过测量主链酰胺基团中（15）N和（1）H之间的剩余偶极耦合，研究了脱辅基肌红蛋白未折叠状态的构象倾向。在拉伸和压缩的聚丙烯酰胺凝胶作用下，脱辅基肌红蛋白在酸性和尿素诱导的未折叠状态下产生了弱排列。在pH 2.3的8 M尿素溶液中，脱辅基肌红蛋白不存在可检测到的二级或三级结构，所有残基均观察到具有统一符号的显著剩余偶极耦合。在pH 2.3且不存在尿素的情况下，多肽局部区域的剩余偶极耦合的大小和/或符号发生变化，这些区域具有形成螺旋二级结构的高倾向。这些结果是基于未折叠多肽链的统计特性来解释的，该多肽链被视为统计片段的聚合物。对于折叠的蛋白质，剩余偶极耦合的大小和符号取决于每个键向量相对于整个分子排列张量的方向，整个分子作为一个单一实体重新定向。对于未折叠的蛋白质，不存在全局排列张量；相反，剩余偶极耦合归因于统计片段或二级结构的瞬态元素的排列。对于8 M尿素中的脱辅基肌红蛋白，主链高度伸展，φ和ψ二面角有利于β或P（II）区域。每个统计片段具有高度各向异性的形状，N-H键向量大致垂直于长轴，并在应变聚丙烯酰胺凝胶的各向异性环境中产生弱排列。剩余偶极耦合大小的降低表征了局部区域增强的柔韧性或链压缩。脱辅基肌红蛋白酸性变性状态下少量螺旋结构的形成导致多肽局部区域剩余偶极耦合符号的变化；根据剩余偶极耦合估计的螺旋数量与根据化学位移确定的数量非常一致。这里描述的脱辅基肌红蛋白的排列模型也可以解释先前报道的葡萄球菌核酸酶和其他蛋白质变性状态的剩余偶极耦合模式。与其他核磁共振实验相结合，剩余偶极耦合可以为蛋白质未折叠和部分折叠状态的动态构象倾向提供有价值的见解，从而有助于描绘折叠景观的上游区域。
