Ratner V, Amir D, Kahana E, Haas E
Faculty of Life Sciences, Bar Ilan University, 52900 Ramat Gan, Israel.
J Mol Biol. 2005 Sep 23;352(3):683-99. doi: 10.1016/j.jmb.2005.06.074.
The various models proposed for protein folding transition differ in their order of appearance of the basic steps during this process. In this study, steady state and time-resolved dynamic non-radiative excitation energy transfer (FRET and trFRET) combined with site specific labeling experiments were applied in order to characterize the initial transient ensemble of Escherichia coli adenylate kinase (AK) molecules upon shifting conditions from those favoring denaturation to refolding and from folding to denaturing. Three sets of labeled AK mutants were prepared, which were designed to probe the equilibrium and transient distributions of intramolecular segmental end-to-end distances. A 176 residue section (residues 28-203), which spans most of the 214 residue molecule, and two short secondary structure chain segments including an alpha-helix (residues 169-188) and a predominantly beta-strand region (residues 188-203), were labeled. Upon fast change of conditions from denaturing to folding, the end-to-end distance of the 176 residue chain section showed an immediate collapse to a mean value of 26 A. Under the same conditions, the two short secondary structure elements did not respond to this shift within the first ten milliseconds, and retained the characteristics of a fully unfolded state. Within the first 10 ms after changes of the solvent from folding to denaturing, only minor changes were observed at the local environments of residues 203 and 169. The response of these same local environments to the shift of conditions from denaturing to folding occurred within the dead time of the mixing device. Thus, the response of the CORE domain of AK to fast transfer from folding to unfolding conditions is slow at all three conformational levels that were probed, and for at least a few milliseconds the ensemble of folded molecules is maintained under unfolding conditions. A different order of the changes was observed upon initiation of refolding. The AK molecules undergo fast collapse to an ensemble of compact structures where the local environment of surface probes seems to be native-like but the two labeled secondary structure elements remain unfolded.
针对蛋白质折叠转变提出的各种模型，在此过程中基本步骤出现的顺序有所不同。在本研究中，稳态和时间分辨动态非辐射激发能量转移（荧光共振能量转移和时间分辨荧光共振能量转移）与位点特异性标记实验相结合，用于表征大肠杆菌腺苷酸激酶（AK）分子在条件从有利于变性转变为复性以及从复性转变为变性时的初始瞬态集合。制备了三组标记的AK突变体，旨在探测分子内片段端到端距离的平衡和瞬态分布。标记了一个跨越214个残基分子大部分的176个残基片段（残基28 - 203），以及两个短的二级结构链段，包括一个α - 螺旋（残基169 - 188）和一个主要为β - 链区域（残基188 - 203）。当条件从变性快速转变为复性时，176个残基链段的端到端距离立即坍缩至平均值26 Å。在相同条件下，两个短的二级结构元件在前十毫秒内对这种转变没有反应，并保持完全未折叠状态的特征。在溶剂从复性转变为变性后的前10毫秒内，仅在残基203和169的局部环境中观察到微小变化。这些相同局部环境对条件从变性转变为复性的响应发生在混合装置的死时间内。因此，在探测的所有三个构象水平上，AK核心结构域对从复性到变性条件的快速转变的响应都很缓慢，并且在变性条件下，折叠分子的集合至少在几毫秒内得以维持。在复性开始时观察到了不同的变化顺序。AK分子快速坍缩为一组紧密结构，其中表面探针的局部环境似乎类似天然状态，但两个标记的二级结构元件仍未折叠。
