Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-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 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.
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.
D M
DM
Gakamsky
D M
DM
Davis
E
E
Haas
J L
JL
Strominger
I
I
Pecht
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D019281:Dimerization; D005456:Fluorescent Dyes; D006183:H-2 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064532:Histocompatibility Antigen H-2D; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014966:Xanthenes; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D006183:H-2 Antigens; D064532:Histocompatibility Antigen H-2D; D015395:Histocompatibility Antigens Class I; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014966:Xanthenes; D001613:beta 2-Microglobulin; C034657:Texas red