Radford S E, Buck M, Topping K D, Dobson C M, Evans P A
Inorganic Chemistry Laboratory, University of Oxford, England.
Proteins. 1992 Oct;14(2):237-48. doi: 10.1002/prot.340140210.
The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.
通过二维核磁共振方法，在pH 7.5和30摄氏度条件下，测定了天然状态下鸡溶菌酶中68个单个酰胺质子的氢交换动力学。这些质子构成了受保护程度最高的酰胺子集，其交换半衰期比从小模型肽的研究中预期的长约10^5 - 10^7倍。在这些条件下观察到的速率分布在很大程度上可以根据单个酰胺的氢键作用及其与大量溶剂的隔离来解释。还测定了溶菌酶可逆变性状态下的交换速率；在非常温和的条件下（pH 2.0、35摄氏度），通过选择性切割Cys-6-Cys-127二硫键交联（CM6-127溶菌酶）降低天然状态的稳定性，使得这一测定成为可能。在这种状态下，大多数酰胺的交换速率在5倍的范围内接近从小模型肽预期的值。然而，对于少数酰胺，有证据表明相对于预测速率有显著延迟（高达近20倍）。在这些条件下观察到的保护模式并不反映蛋白质在强天然条件下的行为，这表明在CM6-127溶菌酶的变性状态下，类似天然结构的区域不会显著持续存在。在高温（pH 3.8、69摄氏度）下天然蛋白质的交换速率模式类似于酸变性状态，这表明在这些条件下，交换动力学主要由瞬时全局展开主导。在这些条件下的折叠和展开速率通过磁化转移核磁共振方法独立测定，从而能够在主要平衡物种为天然状态的条件下，基于该模型推导出变性状态下的固有交换速率。同样，在大多数酰胺的情况下，这些速率与简单随机卷曲模型预测的速率仅显示出有限的偏差。这强化了这样一种观点，即溶菌酶的这些变性状态几乎没有持续的残余秩序，这与蛋白质紧密部分折叠状态的行为形成对比，包括在鸡溶菌酶重折叠过程中瞬时检测到的一种中间体。
