Interaction of Spin Labels with Transition Metals

描述（由申请人提供）：拟议研究的目标是开发，验证和应用电子顺磁共振（EPR）方法来测量非晶体生物系统中的距离。在许多测量距离的技术中，EPR的独特之处在于，顺磁中心可能是天然的，也可能是通过定点突变添加的，电子自旋偶极矩大到足以产生远程相互作用，EPR仅对顺磁中心敏感，甚至深埋在大量的核自旋或吸光物质中。距离信息存在于电子自旋之间的偶极相互作用中，可以用连续波（CW）或脉冲EPR技术来测量。这项提议强调x波段和q波段脉冲技术，因为它们直接测量自旋-自旋相互作用对自旋包的影响，而不需要解释展宽的连续波光谱。q波段提供增强的灵敏度，光谱色散和方向选择。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop, validate, and apply electron paramagnetic resonance (EPR) methodology for measuring distances in non-crystalline biological systems. The unique power of EPR among the many techniques for measuring distances is that the paramagnetic centers may be native or added via site-directed mutagenesis, the electron spin dipole moment is large enough to yield long-range interactions, and EPR is sensitive only to the paramagnetic centers, even deeply buried in vast numbers of nuclear spins or light-absorbing species. The distance information is in the dipolar interaction between electron spins, which can be measured by continuous wave (CW) or pulsed EPR techniques. This proposal emphasizes X-band and Q-band pulse techniques because they measure the impact of spin-spin interaction on spin packets directly, rather than requiring interpretation of broadened CW spectra. Q-band offers enhanced sensitivity, spectral dispersion, and orientation selection. In the current funding period we developed saturation recovery and spin echo methods for measuring distances between rapidly-relaxing heme Fe(III) and a nitroxyl radical. In the proposed funding period we will address unanswered questions related to these techniques and extend them to other iron centers - the iron-sulfur cluster in electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) and the non-heme iron in iron enterobactin (FeEnt) bound to iron protein A (FepA). These systems provide the opportunity to examine the effect of electron spin delocalization (ETF-QO) on the distance measurements and the effect of iron energy level splittings comparable to the EPR quantum (FepA). Pulsed double electron-electron resonance (DEER) is a complementary method to measure distances between two slowly relaxing paramagnetic centers, and we propose to enhance its utility for biological samples. Two inter-related themes extend across the three specific aims - orientation selection and conformational flexibility. The methods that we develop will permit us to address important biological questions raised by our collaborators. We will determine the conformational change in the tonB box of iron protein A, characterize structure changes in the redox active sites of electron transfer flavoprotein ubiquinone reductase, and elucidate the mechanism of DNA melting by large tumor antigen.