Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR
Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR
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DOI：
10.1016/j.jmb.2003.11.042
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发表时间：
2004-01-30
影响因子：
5.6
通讯作者：
Gronenborn, AM
中科院分区：
文献类型：
--
作者：
Ding, KY;Louis, JM;Gronenborn, AM
理解蛋白质的稳定性需要表征折叠和未折叠状态的结构决定因素。许多蛋白质能够在特定的溶液条件下填充部分折叠状态。偶尔，在非或轻度变性条件下，折叠和未折叠状态的共存可以通过NMR观察到，使我们能够在相同条件下从结构上探测这些状态。在这里，我们报告的蛋白G（GB 1）的B1结构域的不稳定突变体的平衡展开进行了系统的研究。测定了不同pH值和不同温度下主链酰胺残基偶极偶联（RDC）、色氨酸NE-H共振和酰胺氮横向弛豫速率（R（2）s）。主链酰胺RDC;表明在完全解折叠之前，在T11、L12和K13周围的转角处以及在A24和T25处的螺旋的N末端处形成两个解链热点。低pH值，热解折叠状态的GB 1的RDC是非常小的，并不表明任何天然样结构的存在。酰胺氮的横向弛豫速率GB 1在折叠状态下，在不同的温度下表现出很大的贡献，从交换过程和相关的动力学显示相当大的异质性。我们的数据提供了明确的证据，中间构象和多态平衡/折叠的GB 1变体。爱思唯尔有限公司出版
Understanding protein stability requires characterization of structural determinants of the folded and unfolded states. Many proteins are capable of populating partially folded states under specific solution conditions. Occasionally, coexistence of the folded and an unfolded state under non- or mildly denaturing conditions can be observed by NMR, allowing us to structurally probe these states under identical conditions. Here we report on a destabilized mutant of the B1 domain of protein G (GB1) whose equilibrium unfolding was systematically investigated. Backbone amide residual dipolar couplings (RDCs), the tryptophan NE-H resonance and the amide nitrogen transverse relaxation rates (R(2)s) for varying pH values and different temperatures were measured. The backbone amide RDCs; indicate that prior to complete unfolding, two melting hot spots are formed at the turn around T11, L12 and K13 and the N terminus of the helix at A24 and T25. The RDCs for the low pH, thermally unfolded state of GB1 are very small and do not indicate the presence of any native-like structure. Amide nitrogen transverse relaxation rates for GB1 in the folded state at different temperatures exhibit large contributions from exchange processes and the associated dynamics display considerable heterogeneity. Our data provide clear evidence for intermediate conformations and multi-state equilibrium un/folding for this GB1 variant. Published by Elsevier Ltd.