Real-time and equilibrium 19F-NMR studies reveal the role of domain-domain interactions in the folding of the chaperone PapD
Real-time and equilibrium 19F-NMR studies reveal the role of domain-domain interactions in the folding of the chaperone PapD
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DOI：
10.1073/pnas.022649599
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发表时间：
2002-01-22
影响因子：
11.1
通讯作者：
Frieden, C
中科院分区：
文献类型：
--
作者：
Bann, JG;Pinkner, J;Frieden, C
PapD是肾盂肾炎大肠杆菌P菌毛形成所必需的周质分子伴侣。杆菌它由两个结构域组成，每个结构域含有一个色氨酸残基（Trp-36和Trp-128，分别位于N端和C端结构域）。为了探索折叠过程中结构域-结构域相互作用的作用，用6-氟色氨酸标记蛋白质用于F-19-NMR实验。收集的F-19-NMR数据作为尿素浓度的函数，揭示了由Trp-128引起的共振的存在，该共振不同于折叠或未折叠的共振。通过停流荧光、CID和F-19-NMR监测从尿素重折叠的时间过程，每种方法显示多个动力学阶段。用氟冷冻探针在70 μ M蛋白质处收集的F-19-NMR停流光谱表明，中间体在折叠过程的早期（< 5 s）被填充。中间共振和未折叠共振的缓慢消失与两个结构域的固有共振的出现以相同的速率发生。数据是一致的模型，其中的C-末端结构域迅速崩溃的中间体，而最终结构的稳定是缓慢的，需要折叠的N-末端结构域与伴随的C-末端结构域结构的重新调整。
PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. coli. It is composed of two domains, each of which contains a tryptophan residue (Trp-36 and Trp-128, in the N- and C-terminal domains, respectively). To explore the role of domain-domain interactions during folding, the protein was labeled with 6-fluorotryptophan for use in F-19-NMR experiments. F-19-NMR data collected as a function of urea concentration revealed the presence of a resonance caused by Trp-128 that was distinct from either the folded or unfolded resonances. The time course of refolding from urea was monitored by stopped-flow fluorescence, CID, and F-19-NMR, each method showing multiple kinetic phases. The F-19-NMR stopped-flow spectra, collected at 70 muM of protein with a fluorine cryoprobe, demonstrated that the intermediate was populated early in the folding process (< 5 s). The slow disappearance of the intermediate and unfolded resonance occurred at the same rate as the appearance of the native resonances of both domains. The data are consistent with a model in which the C-terminal domain collapses rapidly to an intermediate, whereas the stabilization of the final structure is slow and requires folding of the N-terminal domain with concomitant readjustment of the C-terminal domain structure.