Mok K Hun, Kuhn Lars T, Goez Martin, Day Iain J, Lin Jasper C, Andersen Niels H, Hore P J
Department of Chemistry, University of Oxford, Physical & Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, UK.
Nature. 2007 May 3;447(7140):106-9. doi: 10.1038/nature05728. Epub 2007 Apr 11.
Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations. Even though some unfolded and partially folded proteins are now known to possess biological function or to be involved in aggregation phenomena associated with disease states, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR pulse-labelling experiments that involve rapid in situ protein refolding. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.
通过将实验研究与计算机模拟巧妙结合，人们对多肽链在其自由能景观中的构象转变有了深入了解。尽管现在已知一些未折叠和部分折叠的蛋白质具有生物学功能或参与与疾病状态相关的聚集现象，但由于构象异质性和动力学，从实验中获得的关于这些结构的原子水平信息仍然稀少。在此，我们提出一种能够提供此类信息的技术。利用一种名为TC5b的“色氨酸笼”微型蛋白质（参考文献5），我们报告了光化学诱导动态核极化核磁共振脉冲标记实验，该实验涉及快速原位蛋白质重折叠。这些实验使得在未折叠状态下与超极化芳香族侧链核的偶极交叉弛豫能够在所得的折叠态光谱中被识别和量化。我们发现，这种小蛋白质的未折叠状态因疏水塌缩而存在残余结构，在天然状态下彼此相对较远的侧链之间存在强烈的残基间接触。即使形成的是非天然而非天然接触，预先形成结构可能是与蛋白质快速折叠事件相关的一个特征。
