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。在众多测距技术中，EPR的独特之处在于，顺磁中心可以是天然的或通过定点突变添加的，电子自旋偶极矩足够大，足以产生远程相互作用，EPR只对顺磁中心敏感，甚至深埋在大量的核自旋或光吸收物种中。距离信息是电子自旋之间的偶极相互作用，可以用连续波(CW)或脉冲EPR技术测量。这一建议强调了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，而不需要解释加宽的连续波谱。Q波段提供更高的灵敏度、光谱色散和方向选择。 在目前的资助期间，我们开发了饱和恢复和自旋回声方法来测量快速松弛的血红素Fe(III)和氮氧基之间的距离。在拟议的资助期内，我们将解决与这些技术相关的悬而未决的问题，并将其扩展到其他铁中心-电子转移黄素蛋白泛醌氧化还原酶(ETF-QO)中的铁硫簇和与铁蛋白A(FepA)结合的铁肠蛋白中的非血红素铁(FeEnt)。这些系统提供了机会来研究电子自旋离域(ETF-QO)对距离测量的影响，以及与EPR量子(FEPA)相当的铁能级分裂的影响。脉冲双电子-电子共振(DER)是测量两个缓慢弛豫的顺磁中心之间距离的一种互补方法，我们建议加强它在生物样品中的应用。两个相互关联的主题贯穿了三个具体目标--定向选择和构象灵活性。我们开发的方法将使我们能够解决我们的合作者提出的重要生物学问题。我们将测定铁蛋白A的TonB盒的构象变化，表征电子转移黄素蛋白泛醌还原酶氧化还原活性部位的结构变化，并阐明大肿瘤抗原融化DNA的机制。

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.