Structure and dynamics of an acid-denatured protein G mutant.
Structure and dynamics of an acid-denatured protein G mutant.
复制标题
酸变性蛋白 G 突变体的结构和动力学。
作者：
Sari,N;Alexander,P;Bryan,PN;Orban,J
蛋白质变性状态的核磁共振研究提供了对折叠的潜在起始位点的见解，这些位点可能太过短暂而无法进行动力学观察。我们利用gb1的F30H突变体表征了G蛋白酸变性状态的结构和动力学，该突变体处于稳定的边缘。在5°C时，f30h - gb1在pH 7.0时折叠率大于95%，在pH 4.0时未折叠率大于95%。这种稳定性范围是有用的，因为变性状态可以在相对温和的条件下进行检查，这是折叠GB1的最佳条件。几乎所有的15n、HN和h α骨干共振都在酸变性态。化学位移、耦合常数和NOE数据表明，在这些温和条件下研究的变性态比在化学变性剂的存在下有更多的残余结构。酸变性态填充了具有α-螺旋和β-发夹特征的天然构象。据我们所知，这是具有NOE和耦合常数的β-发夹特征的变性态的第一个例子。在折叠态β1 - β2发夹对应的区域，还发现了一些非天然的转弯结构。稳态{1H−15N} NOE结果表明，在变性蛋白的更结构化区域，主链的柔韧性受到限制。总的来说，我们的研究表明，螺旋区域、β3 - β4发夹和β1 - β2转可能是GB折叠的潜在起始位点。此外，酸变性f30h - gb1的残余结构比β1 - β2、α-螺旋和β3 - β4区域对应的肽片段更广泛，表明全长多肽链中存在额外的中远距离相互作用。
NMR studies of protein denatured states provide insights into potential initiation sites for folding that may be too transient to be observed kinetically. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of GB1which is on the margin of stability. At 5 °C, F30H-GB1is greater than 95% folded at pH 7.0 and is greater than 95% unfolded at pH 4.0. This range of stability is useful because the denatured state can be examined under relatively mild conditions which are optimal for folding GB1. We have assigned almost all backbone15N, HN, and Hαresonances in the acid-denatured state. Chemical shift, coupling constant, and NOE data indicate that the denatured state has considerably more residual structure when studied under these mild conditions than in the presence of chemical denaturants. The acid-denatured state populates nativelike conformations with both α-helical and β-hairpin characteristics. To our knowledge, this is the first example of a denatured state with NOE and coupling constant evidence for β-hairpin character. A number of non-native turn structures are also detected, particularly in the region corresponding to the β1−β2 hairpin of the folded state. Steady-state {1H−15N} NOE results demonstrate restricted backbone flexibility in more structured regions of the denatured protein. Overall, our studies suggest that regions of the helix, the β3−β4 hairpin, and the β1−β2 turn may serve as potential initiation sites for folding of GB. Furthermore, residual structure in acid-denatured F30H-GB1is more extensive than in peptide fragments corresponding to the β1−β2, α-helix, and β3−β4 regions, suggesting additional medium-to-long-range interactions in the full-length polypeptide chain.