INCREASING THE DISTANCE RANGE AND RESOLUTION IN PULSED DIPOLAR ESR SPECTROSCOPY

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 表示子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 脉冲偶极ESR光谱具有高的能力，通过执行与感兴趣的对象共价连接的自旋标记之间的定点距离测量来确定生物大分子/复合物的结构。为了获得所研究的结构的足够信息，需要短距离和长距离的几个约束。然而，由于需要使用相当长的演化时间，测量长的自旋间距离是一个挑战。这导致信噪比差，结果解释不可靠。增加相位记忆时间Tm可以提高脉冲偶极ESR实验的灵敏度。在足够低的温度和浓度下，电子自旋的主要弛豫机制与质子自旋扩散有关。因此，可以通过适当选择溶剂或进一步通过使用氘代溶剂作为基质来延长Tm。后一种方法成功地应用于测量溶液中生物物体的距离，最大可达7 nm。由于用于制备模拟膜的脂质中的高质子含量，在膜结合状态下的蛋白质的研究中出现了进一步的并发症。因此，迄今为止，在膜环境中测量的最长距离不超过4.5 nm。 在本文中，我们第一次在基质、蛋白质分子和磷脂的背景下用70或高百分比的氘取代氢的系统中进行距离测量。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Pulsed dipolar ESR spectroscopy has high capacity for the determination of the structure of biomacromolecules/complexes by performing site-directed distance measurements between spin-labels that are covalently attached to the object of interest. To gain sufficient information for the structure that is studied, several constrains of both short and long distances are needed. However, it is a challenge to measure long inter-spin distances due to the necessity of considerably longer evolution times to be used. This leads to a poor signal-to-noise ratio and not reliable interpretation of the results. The sensitivity of pulsed dipolar ESR experiments can be improved by the increase of the phase memory time Tm. At sufficient low temperatures and concentrations the main relaxation mechanism for the electron spins is related to proton spin diffusion. Therefore, Tm can be extended by appropriate choice of solvent or further by using deuterated solvents as a matrix. The later approach was successfully applied to measure distances up to 7 nm in biological objects in solution. Further complications appear in studies of proteins in membrane-bound state due to the high proton content in the lipids used to prepare the mimetic membranes. Thus to date the longest distances, measured in membrane context, do not exceed 4.5 nm. Herein, for the first time we conducted distances measurements in systems with 70 or high percentage substitution of deuterium for hydrogen in context of matrix, protein molecule and phospholipids.