Department of Chemistry, University of California, Davis, 95616, Davis, California.
J Fluoresc. 1994 Sep;4(3):217-26. doi: 10.1007/BF01878454.
Phosphorescence and optically detected magnetic resonance (ODMR) measurements have been carried out on the tryptophan (Trp) residues ofEscherichia coli Trp repressor protein (W Rep) and its two single Trp-containing mutants, W19F and W99F. The enhanced resolution afforded by the W19F and W99F mutants allowed us to characterize the triplet state of boundL-Trp corepressor using phosphorescence wavelengt-selected ORMR spectroscopy. We find that at 77 K the 0,0 band peak wavelength ofL-Trp is shifted from 405.5 nm in the aqueous solvent to ca. 410 nm when bound to the corepressor binding site. This red shift of the phosphorescence along with a corresponding increase in the zero-field splittingE value and narrowing of the ODMR linewidth characterize a binding site that is less polar, as well as more polarizable and homogeneous, than the aqueous solvent. This conclusion is in agreement with the X-ray crystallographic structure of the holorepressor protein that places the indole chromophore of the bound corepressor in a cleft in which it is sandwiched by the side chains of arginines 54 and 84.
已对色氨酸（Trp）残基进行了磷光和光探测磁共振（ODMR）测量大肠杆菌色氨酸阻遏蛋白（W Rep）及其两个单色氨酸残基突变体 W19F 和 W99F。W19F 和 W99F 突变体提供的增强分辨率使我们能够使用磷光波长选择 ORMR 光谱来表征结合的 L-Trp 核心抑制剂的三重态。我们发现，在 77 K 时，结合到核心抑制剂结合位点时，L-Trp 的 0,0 带峰波长从水溶液中的 405.5nm 红移至约 410nm。这种磷光的红移以及零场分裂 E 值的相应增加和 ODMR 线宽的变窄，表明结合部位的极性比水溶液小，可极化性和均匀性更高。这一结论与 holorepressor 蛋白的 X 射线晶体结构一致，该结构将结合的核心抑制剂的吲哚发色团置于其中，被精氨酸 54 和 84 的侧链夹在裂缝中。
