Photophysical analysis of class I major histocompatibility complex proteinassembly using a xanthene-derivatized beta(2)-microglobulin

Spectral changes and a sixfold increase in the emission intensity were obse rved in the fluorescence of a single xanthene probe (Texas red) attached to beta(2)m-microglobulin (beta(2)m) upon assembly of beta(2)m into a ternary complex with mouse H-2K(d) heavy chain and influenza nuclear protein pepti de. Dissociation of the labeled beta(2)m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission in tensity. Thus changes in xanthene probe fluorescence upon association/disso ciation of the labeled beta(2)m molecule with/from the ternary complex prov ide a simple and convenient method for studying the assembly/dissociation m echanism of the class I major histocompatibility complex (MHC-I) encoded mo lecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta(2)m molecules. The fluorescence at 610 nm is due to beta(2)m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to th ose of xanthene monomers. Fluorescence around 630 nm is due to beta(2)m oli gomers where xanthene probes interact. Minima in the steady-state excitatio n (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxi ma of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are e xplained by splitting of the first singlet excited state of interacting xan thene probes that can be modeled by exciton theory.