New ways to probe chemical structure & dynamics using multi-frequency pulsed EPR

我提出了一种新的方法，使用时域，多频EPR来确定氮氧化物自旋标签和复杂分子（如聚合物或蛋白质）中快速弛豫顺磁性金属离子之间的距离和方向。这可以通过测量自旋标签在不同场下的纵向弛豫时间T1来实现，该时间通过与快速弛豫各向异性顺磁性金属中心的偶极子-偶极子耦合而增强。该程序将适用于低温和室温条件，也将使自旋标签和分子结构域的复杂运动动力学分析成为可能。利用布朗动力学轨迹的方法对不同类型的自旋标签和分子的运动动力学进行了建模，并设计了通用的计算机模拟程序。验证将通过测量已知结构的化学和生物系统在不同频率下的纵向自旋弛豫时间来进行，以提供自旋标签和快速弛豫金属中心之间的一组距离和角度，包括用于超快速弛豫的镧系（III）离子。与半胱氨酸残基共价连接的镧系络合物可以测定两个不同标记的蛋白质之间的距离角。开发的方法将应用于东英吉利大学的金属生物学中心，在那里安装了布鲁克混合X/ w波段傅里叶变换脉冲EPR仪器，开辟了新的实验机会，这在英国迄今为止是独一无二的。新的方法将为各种系统的应用开辟可能性，从液体和分子晶体、聚合物到膜和整个细胞中的蛋白质。

I propose a novel methodology, using time domain, multi-frequency EPR to determine distances andorientation between a nitroxide spin label and a fast relaxing paramagnetic metal ion within a complex molecule like polymer or protein. This can be achieved by measuring the longitudinal relaxation time, T1 , of a spin label at different fields, which is enhanced by dipole-dipole coupling with the fast relaxing anisotropic paramagnetic metal centre. The procedures will be applicable both at low and room temperature conditions and alsowill enable analysis of complex motional dynamics of both the spin label and a molecular domain. The different types of motional dynamics of spin label and molecules will be modelled using the method of Brownian dynamics trajectories and a general computer simulation program will be designed. Validation will be carried out by measurement of longitudinal spin relaxation times at different frequencies from chemical and biological systems of known structure labelled to provide a set of distances and angels between a spin label and a rapidly relaxing metal centre including lanthanide(III) ions for ultra-fast relaxation. Lanthanide complexes covalently attached to a cystein residue will allow determination of distance-angles between two differently labelled proteins. Methodology developed will be applied at the Metallobiology Centre at UEA where new experimental opportunities have open up with the installation of a Bruker hybrid X/W-band Fourier Transform, pulsed EPR instrument, unique so far in the UK. New methods would open up the possibility for applications to various systems from liquid and molecular crystals and polymers to proteins in membrane and in the whole cell.