High-field EPR spectroscopy applied to biological systems: characterization of molecular switches for electron and ion transfer.
High-field EPR spectroscopy applied to biological systems: characterization of molecular switches for electron and ion transfer.
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高场 EPR 光谱应用于生物系统：电子和离子转移分子开关的表征。
DOI：
10.1039/b412180e
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
M. Fuchst
中科院分区：
文献类型：
--
作者：
K. Möbius;A. Savitsky;A. Schnegg;M. Plato;M. Fuchst
在过去的十年里，生物学家、化学家和物理学家在了解决定蛋白质跨膜转移过程的特异性和方向性的主要因素方面取得了巨大的进展。各种各样的实验技术正在被使用，包括x射线和中子衍射，还有时间分辨光学，红外和磁共振光谱。这是与基因工程策略一起完成的，以构建位点特异性突变体来控制蛋白质的修饰。作为对这些努力的一般认识，认识到蛋白质及其膜界面内的弱相互作用的实质性影响。由于蛋白质-膜复合物固有的柔韧性，弱相互作用在反应周期中会发生微妙的变化。特定的构象变化完成分子开关功能，使转移过程以最佳效率进行。时变非键相互作用的典型例子是特定的h模式和/或微环境的极性效应。目前的认知来自于新开发的光谱技术（在这方面先进的EPR当然值得赞扬）与新开发的计算策略的结合，这些计算策略从蛋白质结构和动力学的角度解释实验数据。到目前为止，这种耦合的合作伙伴，特别是高场EPR光谱和基于dft的量子理论，已经达到了应用于大型生物复合物的复杂程度。本文综述了近年来我们实验室研究的几个大型范式生物系统。与传统的x波段EPR （9.5 GHz/0.34 T）相比，利用95 GHz/3.4 T和360 GHz/12.9 T下高频/高场EPR提高的光谱和时间分辨率，对三个生物系统进行了结构和动力学表征：(1)光合细菌球形红杆菌（Rhodobacter sphaeroides）野生型和突变型反应中心蛋白的光诱导电子转移中间体，(2)盐杆菌（Halobacterium salinarium）细菌紫质蛋白位点特异性氮氧自旋标记突变体的光驱动质子转移中间体，(3)大肠杆菌（Escherichia coli）细菌毒素Colicin A通道形成蛋白域的位点特异性氮氧自旋标记突变体的重折叠中间体。所获得的详细信息是对蛋白质晶体学、固态核磁共振、红外和光学光谱技术的补充。高场EPR的独特强度特别值得注意：它可以提供生物作用中蛋白质瞬态中间体的高度所需的详细信息。它们可以在生物学相关的时间尺度上保持工作状态时进行观察和表征。这篇综述向读者介绍了与核磁共振相关的EPR的起源和基本实验，描述了将传统EPR扩展到高场/高频EPR的基本策略，并强调了从高场EPR与基因工程结合获得的分子信息的细节，这些细节是“经典”光谱学无法获得的。强调了用DFT和先进的半经验分子轨道理论对实验数据进行量子化学解释的重要性。简要介绍了柏林大学实验室研制的95 GHz和360 GHz EPR/ENDOR光谱仪。最后，对先进生物epr在跨学科研究中的机遇和挑战进行了展望。
The last decade witnessed a tremendous growth in combined efforts of biologists, chemists and physicists to understand the dominant factors determining the specificity and directionality of transmembrane transfer processes in proteins. A large variety of experimental techniques is being used including X-ray and neutron diffraction, but also time-resolved optical, infrared and magnetic resonance spectroscopy. This is done in conjunction with genetic engineering strategies to construct site-specific mutants for controlled modification of the proteins. As a general perception of these efforts, the substantial influence of weak interactions within the protein and its membrane interfaces is recognized. The weak interactions are subject to subtle changes during the reaction cycle owing to the inherent flexibility of the protein-membrane complex. Specific conformational changes accomplish molecular-switch functions for the transfer process to proceed with optimum efficiency. Characteristic examples of time varying non-bonded interactions are specific H-patterns and/or polarity effects of the microenvironment. The present perception has emerged from the coupling of newly developed spectroscopic techniques - and advanced EPR certainly deserves credit in this respect - with newly developed computational strategies to interpret the experimental data in terms of protein structure and dynamics. By now, the partners of this coupling, particularly high-field EPR spectroscopy and DFT-based quantum theory, have reached a level of sophistication that applications to large biocomplexes are within reach. In this review, a few large paradigm biosystems are surveyed which were explored lately in our laboratory. Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), three biosystems are characterized with respect to structure and dynamics: (1) Light-induced electron-transfer intermediates in wild-type and mutant reaction-centre proteins from the photosynthetic bacterium Rhodobacter sphaeroides, (2) light-driven proton-transfer intermediates of site-specifically nitroxide spin-labelled mutants of bacteriorhodopsin proteins from Halobacterium salinarium, (3) refolding intermediates of site-specifically nitroxide spin-labelled mutants of the channel-forming protein domain of Colicin A bacterial toxin produced in Escherichia coli. The detailed information obtained is complementary to that of protein crystallography, solid-state NMR, infrared and optical spectroscopy techniques. A unique strength of high-field EPR is particularly noteworthy: it can provide highly desired detailed information on transient intermediates of proteins in biological action. They can be observed and characterized while staying in their working states on biologically relevant time scales. The review introduces the audience to origins and basic experiments of EPR in relation to NMR, describes the underlying strategies for extending conventional EPR to high-field/high-frequency EPR, and highlights those details of molecular information that are obtained from high-field EPR in conjunction with genetic engineering and that are not accessible by "classical" spectroscopy. The importance of quantum-chemical interpretation of the experimental data by DFT and advanced semiempirical molecular-orbital theory is emphasized. A short description of the laboratory-built 95 GHz and 360 GHz EPR/ENDOR spectrometers at FU Berlin is also presented. The review concludes with an outlook to future opportunities and challenges of advanced bio-EPR in interdisciplinary research.
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影响因子：
56.9
作者：
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通讯作者：
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DOI：
10.1073/pnas.81.24.7792
发表时间：
1984
影响因子：
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影响因子：
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作者：
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通讯作者：
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DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
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作者：
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通讯作者：
Hermodson,M