Bridges Michael D, Hideg Kálmán, Hubbell Wayne L
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008, USA.
Appl Magn Reson. 2010 Jan 1;37(1-4):363. doi: 10.1007/s00723-009-0079-2.
The function of many proteins involves equilibria between conformational substates, and to elucidate mechanisms of function it is essential to have experimental tools to detect the presence of conformational substates and to determine the time scale of exchange between them. Site-directed spin labeling (SDSL) has the potential to serve this purpose. In proteins containing a nitroxide side chain (R1), multicomponent electron paramagnetic resonance (EPR) spectra can arise either from equilibria involving different conformational substates or rotamers of R1. To employ SDSL to uniquely identify conformational equilibria, it is thus essential to distinguish between these origins of multicomponent spectra. Here we show that this is possible based on the time scale for exchange of the nitroxide between distinct environments that give rise to multicomponent EPR spectra; rotamer exchange for R1 lies in the ≈0.1-1 μs range, while conformational exchange is at least an order of magnitude slower. The time scales of exchange events are determined by saturation recovery EPR, and in favorable cases, the exchange rate constants between substates with lifetimes of approximately 1-70 μs can be estimated by the approach.
许多蛋白质的功能涉及构象亚态之间的平衡，为了阐明功能机制，拥有检测构象亚态的存在并确定它们之间交换时间尺度的实验工具至关重要。定点自旋标记（SDSL）有潜力用于此目的。在含有氮氧化物侧链（R1）的蛋白质中，多组分电子顺磁共振（EPR）光谱可能源于涉及不同构象亚态或R1旋转异构体的平衡。为了利用SDSL唯一地识别构象平衡，因此必须区分多组分光谱的这些来源。在这里我们表明，基于产生多组分EPR光谱的不同环境之间氮氧化物交换的时间尺度，这是可能的；R1的旋转异构体交换在≈0.1 - 1微秒范围内，而构象交换至少慢一个数量级。交换事件的时间尺度由饱和恢复EPR确定，在有利的情况下，通过该方法可以估计寿命约为1 - 70微秒的亚态之间的交换速率常数。
