Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized β2-microglobulin

Spectral changes and a sixfold increase in the emission intensity were observed 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 peptide. Dissociation of the labeled beta(2)m 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 beta(2)m 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 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 those of xanthene monomers. Fluorescence around 630 nm is due to beta(2)m 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.