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

Dmitry M. Gakamsky, Daniel M. Davis, Elisha Haas, Jack L. Strominger, Israel Pecht

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Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to β2m-microglobulin (β2m) upon assembly of β2m into a ternary complex with mouse H-2K(d) heavy chain and influenza nuclear protein peptide. Dissociation of the labeled β2m 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 β2m 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 β2m molecules. The fluorescence at 610 nm is due to β2m 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 β2m 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.

Original languageEnglish
Pages (from-to)1552-1560
Number of pages9
JournalBiophysical Journal
Issue number3
StatePublished - Mar 1999

Bibliographical note

Funding Information:
This study was supported by the European Community, grant B104-CT96-0135, and by National Institutes of Health research grant CA-45774. DMD acknowledges financial support from a postdoctoral fellowship awarded by the Irvington Institute. K d /human β 2 -microglobulin heterodimer was a kind gift from Dr. Siegfrid Weiss.


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