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首页> 外文学位 >Mechanistic Investigations on Hsp70 Chaperone-Mediated Protein Folding, and, Photo-CIDNP enhancements in Heteronuclear Correlation NMR Spectroscopy.
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Mechanistic Investigations on Hsp70 Chaperone-Mediated Protein Folding, and, Photo-CIDNP enhancements in Heteronuclear Correlation NMR Spectroscopy.

机译：Hsp70分子伴侣介导的蛋白质折叠和Photo-CIDNP增强在异核相关NMR光谱中的机理研究。

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摘要

Part1: The Hsp70 family of molecular chaperones is ubiquitous and highly conserved. The Hsp70 chaperone system participates in a remarkable variety of cellular processes, including co- and post-translational protein folding. Hsp70 associates with its client proteins in an ATP- regulated manner. However, the molecular role of chaperone-substrate interactions in enabling 1isp70 function remains unclear.;We have integrated stopped-flow fast-mixing, kinetic modeling and multidimensional NMR to investigate mechanisms of the E. coli Hsp70 chaperone system (comprising DnaK, DnaJ and GrpE, referred to as K/J/E). Our kinetic simulations show that the kinetics of the K/J/E chaperone system is optimized to utilize protein folding rates as a substrate selectivity mechanism. The experimentally assessed interaction of E. coli RNase H D with K/J/E during folding is consistent with a kinetic partitioning model of chaperone-substrate interactions. DnaK interacts extensively but transiently with RNase HD, thereby reversibly sequestering non-native client molecules, while concurrently avoiding substantial delays in RNase HD folding. NMR studies show that the substrate conformation is substantially altered upon DnaK-binding and that DnaK-bound substrates assume multiple structures. However, the substrate conformation remains unaffected by the DnaK nucleotide state.;Part2: NMR is an outstanding biophysical tool and offers atomic resolution structural and dynamic information. The applicability of solution NMR methodology to complex biochemical systems has been limited by the low NMR sensitivity. Photochemically induced dynamic nuclear polarization (photo-CIDNP) is the only hyperpolarization method with demonstrated applicability to solution state protein NMR.;Here, we explore the potential of 1H, 15N and 13C photo-CIDNP to enhance the sensitivity of heteronuclear correlation NMR. We report that the HPE-SOFAST-HMQC is the most sensitive 1H photo-CIDNP-enhanced (HPE) pulse sequence. We have developed two new pulse sequences, EPIC- and CHANCE-HSQC, designed to optimally utilize photo-CIDNP sensitivity enhancements from heteronuclei such as 15N and 13C. EPIC- and CHANCE-HSQC are amenable to 2D data acquisition, and sensitivity improvements arising from these two sequences are modeled effectively using standard photo-CIDNP rate equations. We observe backbone 13C nuclei to be photo-CIDNP polarizable, enabling the high sensitivity evaluation of secondary structure. 13C photo-CIDNP provides large (up to 16-fold) sensitivity enhancements compared to the conventional HSQC pulse sequence.
机译：第一部分：Hsp70分子伴侣家族无处不在，并且高度保守。 Hsp70分子伴侣系统参与各种细胞过程，包括共翻译和翻译后蛋白质折叠。 Hsp70以ATP调节的方式与其客户蛋白质结合。然而，分子伴侣-底物相互作用在启用1isp70功能中的分子作用尚不清楚。;我们已经集成了停止流快速混合，动力学建模和多维NMR，以研究大肠杆菌Hsp70分子伴侣系统（包括DnaK，DnaJ和GrpE，称为K / J / E）。我们的动力学模拟表明，K / J / E分子伴侣系统的动力学经过优化，可利用蛋白质折叠速率作为底物选择性机制。折叠过程中大肠杆菌RNase H D与K / J / E相互作用的实验评估与分子伴侣-底物相互作用的动力学分配模型相符。 DnaK与RNase HD广泛但短暂地相互作用，从而可逆地隔离非本地客户分子，同时避免了RNase HD折叠的实质性延迟。 NMR研究表明，底物构象在DnaK结合后会发生实质性改变，并且DnaK结合的底物呈现多种结构。然而，底物构象仍然不受DnaK核苷酸状态的影响。；第二部分：NMR是一种出色的生物物理工具，可提供原子分辨率的结构和动态信息。溶液NMR方法在复杂的生化系统中的适用性受到NMR灵敏度低的限制。光化学诱导的动态核极化（photo-CIDNP）是唯一证明对溶液态蛋白NMR具有适用性的超极化方法。在此，我们探索1H，15N和13C photo-CIDNP增强异核相关NMR灵敏度的潜力。我们报告说，HPE-SOFAST-HMQC是最敏感的1H光CIDNP增强（HPE）脉冲序列。我们已经开发了两个新的脉冲序列，EPIC和CHANCE-HSQC，旨在最佳利用来自15N和13C等杂核的photo-CIDNP灵敏度增强。 EPIC和CHANCE-HSQC适合2D数据采集，并且使用标准的photo-CIDNP速率方程可有效地对这两个序列产生的灵敏度提高进行建模。我们观察到骨架13C核是光CIDNP可极化的，从而能够对二级结构进行高灵敏度评估。与传统的HSQC脉冲序列相比，13C photo-CIDNP可以提供较大的灵敏度提高（最多16倍）。

著录项

  • 作者

    Sekhar, Ashok.;

  • 作者单位

    The University of Wisconsin - Madison.;

  • 授予单位 The University of Wisconsin - Madison.;
  • 学科 Chemistry Biochemistry.;Biophysics General.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 411 p.
  • 总页数 411
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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