Gakamsky D M, Davis D M, Haas E, Strominger J L, Pecht I
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.
Biophys J. 1999 Mar;76(3):1552-60. doi: 10.1016/S0006-3495(99)77314-5.
Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory.
当β2m与小鼠H-2Kd重链和流感核蛋白肽组装形成三元复合物时，观察到附着在β2m上的单个呫吨探针（德克萨斯红）的荧光发生光谱变化，发射强度增加了六倍。标记的β2m从三元复合物中解离恢复了探针的荧光和吸收光谱，并降低了发射强度。因此，标记的β2m分子与三元复合物结合/解离时呫吨探针荧光的变化为研究I类主要组织相容性复合体（MHC-I）编码分子的组装/解离机制提供了一种简单便捷的方法。探针的光物理变化可以用游离标记的β2m分子的寡聚化来解释。610nm处的荧光归因于β2m二聚体，其中探针在空间上显著分离，因此它们的发射和激发特性接近呫吨单体。630nm左右的荧光归因于呫吨探针相互作用的β2m寡聚体。稳态激发（550nm）和发射（630nm）各向异性光谱的最小值与高阶寡聚体激发和发射光谱的最大值相关，表明它们的荧光去极化程度更高。这些光物理特征可以通过相互作用的呫吨探针的第一单线态激发态的分裂来解释，这可以用激子理论来建模。
