MULTIFREQUENCY ESR STUDIES OF PROTEIN DYNAMICS T4 LYSOZYME

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 结合高和低ESR频率的多频率方法可以通过位点定向自旋标记(SDSL)来分解影响蛋白质标记上每个位点标记的复杂动态模式。在最近的一项利用诱变剂对T4溶菌酶的研究中，我们发现高场/高频ESR(HFHF-ESR)实验可以满意地用微观有序宏观无序(MOMD)模型来描述，在该模型中只存在内部动力学。缓慢松弛局部结构(SRLS)模型包括对整体滚动率和内部模式的拟合参数。MOMD模型是SRLS模型的一种极限情况，其中整体滚流速度太慢，不会影响光谱的外观。在9 GHz频谱较慢的时间尺度上，较快的内模和较慢的整体滚落都是重要的。这些研究为多频ESR绘制大分子逐个位置的动态结构的潜力提供了证据。该中心最近安装了新的HFHF-ESR CW光谱仪，与以前的版本相比，它具有更好的信噪比和温度稳定性，这将使我们能够研究系统的动态模式，在9到240 GHz的频率范围内提高了灵敏度和吞吐能力。频谱的可用性可用于分析一系列频率，尽管这是一个巨大的计算挑战，但将为系统的动态模式提供详细的见解。对我们最初在95 GHz开发的所有谐振器实施连续可变耦合将是进一步提高我们高频ESR光谱仪灵敏度的关键进步。

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. A multifrequency approach combining high and low ESR frequencies can enable a decomposition of the complex dynamic modes affecting each site label on a protein labeled via site-directed spin-labeling (SDSL). In a recent study on T4 lysozyme using mutagens, to which a spin label is attached at different residues, we showed that the high-field/high-frequency-ESR (HFHF-ESR) experiments could be satisfactorily described by the microscopic order macroscopic disorder (MOMD) model in which only the internal dynamics is present. The slowly relaxing local structure (SRLS) model includes fitting parameters for the overall tumbling rates and internal modes. The MOMD model is a limiting case of SRLS model where the overall tumbling is too slow to affect the appearance of the spectrum. On the slower time scale of the 9GHz spectra both the faster internal modes and the slower overall tumbling are important. These studies have provided evidence for the potential of multifrequency ESR to map out the dynamic structure, on a site to site basis, of a macromolecule. The center has recently installed new HFHF-ESR cw spectrometers with superior signal to noise and temperature stability to previous versions, which will allow us to study the dynamic modes of the system with improved sensitivity and throughput capability over a range of frequencies from 9 to 240GHz. The availability of spectra for analysis over a range of frequencies, although a significant computational challenge, will provide detailed insights into the dynamic modes of the system. The implementation of continuously variable coupling to all of our resonators originally developed at 95GHz will be a crucial advance to further improve the sensitivity of our HFHF ESR spectrometers.