Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1999 Mar;76(3):1552–1560. doi: 10.1016/S0006-3495(99)77314-5

Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin.

D M Gakamsky 1, D M Davis 1, E Haas 1, J L Strominger 1, I Pecht 1
PMCID: PMC1300131  PMID: 10049335

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (130.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Davis D. M., Reyburn H. T., Pazmany L., Chiu I., Mandelboim O., Strominger J. L. Impaired spontaneous endocytosis of HLA-G. Eur J Immunol. 1997 Oct;27(10):2714–2719. doi: 10.1002/eji.1830271035. [DOI] [PubMed] [Google Scholar]
  2. Demaria S., Schwab R., Bushkin Y. The origin and fate of beta 2m-free MHC class I molecules induced on activated T cells. Cell Immunol. 1992 Jun;142(1):103–113. doi: 10.1016/0008-8749(92)90272-q. [DOI] [PubMed] [Google Scholar]
  3. Edidin M., Achilles S., Zeff R., Wei T. Probing the stability of class I major histocompatibility complex (MHC) molecules on the surface of human cells. Immunogenetics. 1997;46(1):41–45. doi: 10.1007/s002510050240. [DOI] [PubMed] [Google Scholar]
  4. Fahnestock M. L., Tamir I., Narhi L., Bjorkman P. J. Thermal stability comparison of purified empty and peptide-filled forms of a class I MHC molecule. Science. 1992 Dec 4;258(5088):1658–1662. doi: 10.1126/science.1360705. [DOI] [PubMed] [Google Scholar]
  5. Gakamsky D. M., Bjorkman P. J., Pecht I. Peptide interaction with a class I major histocompatibility complex-encoded molecule: allosteric control of the ternary complex stability. Biochemistry. 1996 Nov 26;35(47):14841–14848. doi: 10.1021/bi961707u. [DOI] [PubMed] [Google Scholar]
  6. Gottfried D. S., Haas E. Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor. Biochemistry. 1992 Dec 15;31(49):12353–12362. doi: 10.1021/bi00164a009. [DOI] [PubMed] [Google Scholar]
  7. Hamman B. D., Oleinikov A. V., Jokhadze G. G., Bochkariov D. E., Traut R. R., Jameson D. M. Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers. J Biol Chem. 1996 Mar 29;271(13):7568–7573. doi: 10.1074/jbc.271.13.7568. [DOI] [PubMed] [Google Scholar]
  8. Hyafil F., Strominger J. L. Dissociation and exchange of the beta 2-microglobulin subunit of HLA-A and HLA-B antigens. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5834–5838. doi: 10.1073/pnas.76.11.5834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kasha M. Energy transfer, charge transfer, and proton transfer in molecular composite systems. Basic Life Sci. 1991;58:231–255. doi: 10.1007/978-1-4684-7627-9_8. [DOI] [PubMed] [Google Scholar]
  10. Machy P., Truneh A., Gennaro D., Hoffstein S. Major histocompatibility complex class I molecules internalized via coated pits in T lymphocytes. Nature. 1987 Aug 20;328(6132):724–726. doi: 10.1038/328724a0. [DOI] [PubMed] [Google Scholar]
  11. Osborn C. K., Grigoriev V., Crew M. D. Modulation of class I major histocompatibility complex antigen cell-surface stability by transmembrane domain length variation. Mol Immunol. 1997 Aug;34(11):771–780. doi: 10.1016/s0161-5890(97)00069-2. [DOI] [PubMed] [Google Scholar]
  12. Packard B. Z., Toptygin D. D., Komoriya A., Brand L. Profluorescent protease substrates: intramolecular dimers described by the exciton model. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11640–11645. doi: 10.1073/pnas.93.21.11640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Parker K. C., Strominger J. L. Subunit interactions of class I histocompatibility antigens. Biochemistry. 1985 Sep 24;24(20):5543–5550. doi: 10.1021/bi00341a039. [DOI] [PubMed] [Google Scholar]
  14. Smith J. D., Lie W. R., Gorka J., Myers N. B., Hansen T. H. Extensive peptide ligand exchange by surface class I major histocompatibility complex molecules independent of exogenous beta 2-microglobulin. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7767–7771. doi: 10.1073/pnas.89.16.7767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Townsend A., Elliott T., Cerundolo V., Foster L., Barber B., Tse A. Assembly of MHC class I molecules analyzed in vitro. Cell. 1990 Jul 27;62(2):285–295. doi: 10.1016/0092-8674(90)90366-m. [DOI] [PubMed] [Google Scholar]
  16. Tse D. B., Cantor C. R., McDowell J., Pernis B. Recycling class I MHC antigens: dynamics of internalization, acidification, and ligand-degradation in murine T lymphoblasts. J Mol Cell Immunol. 1986;2(6):315–329. [PubMed] [Google Scholar]
  17. Tse D. B., Pernis B. Spontaneous internalization of Class I major histocompatibility complex molecules in T lymphoid cells. J Exp Med. 1984 Jan 1;159(1):193–207. doi: 10.1084/jem.159.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wahl P., Weber G. Fluorescence depolarization of rabbit gamma globulin conjugates. J Mol Biol. 1967 Dec 14;30(2):371–382. doi: 10.1016/s0022-2836(67)80045-7. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES