Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR
Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR
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DOI：
10.1021/bi048614u
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发表时间：
2004-11-02
期刊：
影响因子：
2.9
通讯作者：
Frieden, C
中科院分区：
文献类型：
--
作者：
Bann, JG;Frieden, C
已经研究了双结构域细菌伴侣PapD的折叠，以了解单个结构域折叠与结构域-结构域相互作用形成之间的关系。PapD含有六个苯丙氨酸残基，四个在N-末端结构域，两个在C-末端结构域。为了检查PapD的折叠特性，用对氟苯丙氨酸（F-19-Phe）均匀地和位点特异性地标记蛋白质，用于F-19 NMR研究，结合圆二色性和荧光研究。在平衡变性实验监测的F-19 NMR，19 F-Phe的天然强度的N-和C-末端结构域的损失显示相同的依赖于尿素浓度。对于N-末端结构域，天然强度的损失通过单独的变性共振的出现来反映。对于C-末端结构域，其中包含残基苯丙氨酸168和苯丙氨酸205，中间以及变性共振出现。这些中间共振坚持在变性剂浓度远远超过损失的天然共振强度，并出现在动力学重折叠F-19 NMR实验。在脯氨酸异构化不影响重折叠动力学的双跳F-19 NMR实验中，如果蛋白质仅短时间变性，则结构域-结构域相互作用的形成是快速的。然而，随着变性时间的增加，N-和C-末端结构域的天然强度降低，并且N-末端结构域的变性共振和C-末端结构域的中间共振累积。的N-末端结构域共振的损失率是一致的顺式到反式异构化过程中，表明从平衡变性状态的缓慢折叠的PapD是由于一个或两个N-末端顺式脯氨酸残基的反式到顺式异构化。数据表明，N-和C-末端结构域必须折叠成天然构象之前形成的结构域-结构域相互作用。
The folding of the two-domain bacterial chaperone PapD has been studied to develop an understanding of the relationship between individual domain folding and the formation of domain-domain interactions. PapD contains six phenylalanine residues, four in the N-terminal domain and two in the C-terminal domain. To examine the folding properties of PapD, the protein was both uniformly and site-specifically labeled with p-fluoro-phenylalanine (F-19-Phe) for F-19 NMR studies, in conjunction with those of circular dichroism and fluorescence. In equilibrium denaturation experiments monitored by F-19 NMR, the loss of 19F-Phe native intensity for both the N- and C-terminal domains shows the same dependence on urea concentration. For the N-terminal domain the loss of native intensity is mirrored by the appearance of separate denatured resonances. For the C-terminal domain, which contains residues Phe 168 and Phe 205, intermediate as well as denatured resonances appear. These intermediate resonances persist at denaturant concentrations well beyond the loss of native resonance intensity and appear in kinetic refolding F-19 NMR experiments. In double-jump F-19 NMR experiments in which proline isomerization does not affect the refolding kinetics, the formation of domain-domain interactions is fast if the protein is denatured for only a short time. However, with increasing time of denaturation the native intensities of the N- and C-terminal domains decrease, and the denatured resonances of the N-terminal domain and the intermediate resonances of the C-terminal domain accumulate. The rate of loss of the N-terminal domain resonances is consistent with a cis to trans isomerization process, indicating that from an equilibrium denatured state the slow refolding of PapD is due to the trans to cis isomerization of one or both of the N-terminal cis proline residues. The data indicate that both the N- and C-terminal domains must fold into a native conformation prior to the formation of domain-domain interactions.