MULTIFREQUENCY STUDIES OF SPIN-LABELED HIV-1 PROTEASE

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。 定点自旋标记(SDSL)是监测可溶性蛋白和膜蛋白结构和动力学的有力工具。用电子顺磁共振(EPR)光谱测量顺磁性氮氧化物探针的光谱性质，为蛋白质中的环境提供了丰富的信息。HIV-1蛋白酶是一种22 kDa的同源二聚体蛋白，对艾滋病病毒的功能是必不可少的，而蛋白酶抑制剂已经被开发成有效的HIV药物。我们对HIV-1酶瓣动力学的细节特别感兴趣，这些细节可以从多频率的SDSL EPR研究中了解到。早些时候对9.5 GHz下几个自旋标记的SDSL研究没有显示出在存在或不存在蛋白酶抑制剂的情况下翻盖动力学的任何显著变化，与预期相反。170 GHz的初步结果确实表明，在存在或不存在抑制剂的情况下，襟翼动力学存在微小但可辨别的差异，抑制形式显示出较慢的翻滚的明显证据。基于这些有希望的初步结果，我们正在改进实验设计，以增强光谱差异。我们认为，240 GHz的互补性研究将进一步解决光谱对观察到的动力学微小差异的敏感性，并将为量化HIV-1蛋白酶对各种抑制剂存在的反应提供有用的手段。

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. Site-directed spin labeling (SDSL) is a powerful tool for monitoring the structure and dynamics of both soluble and membrane proteins. Measurements of the spectral properties of the paramagnetic nitroxide probe with electron paramagnetic resonance (EPR) spectroscopy provide a wealth of information on the environment in the protein. The HIV-1 protease is a 22 kDa homodimeric protein essential for function of the AIDS virus, and protease inhibitors have been developed into effective HIV drugs. We are particularly interested in the details of HIV-1 protease flap dynamics that can be learned from multifrequency SDSL EPR studies. Earlier SDSL studies of several spin labels at 9.5 GHz do not reveal any significant change in the flap dynamics in the presence or absence of protease inhibitor, contrary to expectration. Preliminary results at 170 GHz do indicate small but discernible differences in the flap dynamics in the presence or absence of inhibitor with the inhibited form showing clear evidence of slower tumbling. Based on these promising initial results we are refining the experimental design in order to enhance the spectral differences. We suggest that complementary studies at 240 GHz will further resolve the spectral sensitivity to the observed small differences in dynamics and will provide a useful means for quantifying the response of HIV-1 protease to the presence of various inhibitors.