The human class I major histocompatibility complex (MHC) encoded molecule HLA-A2 loaded with the high-affinity peptide GILGRVFTL (p790), was studied by means of steady-state and picosecond fluorescence intensity and fluorescence anisotropy methods. The large number of tryptophan residues (W) (10 W/heavy chain, 2W β2m) as well as their fluorescence sensitivity to the microenvironment, determine the emission of the studied complex. The HLA-A2/peptide complex exhibits a considerable static inhomogeneous broadening (IB) of the W electronic spectra, which results in a dependence of the steady-state fluorescence spectrum on the excitation wavelength. The high concentration of W's chromophores and the spectral IB cause a directed non-radiative migration of electronic excitation energy by Foerster's mechanism from 'blue' W residues to 'red' ones. This phenomenon manifests itself in a nanosecond fluorescence spectral shift and an accelerated fluorescence depolarization at the red edge of the emission spectrum. Selective excitation at the red edge of the W absorption band (310 nm) provided a space selective reduction in the number of excited chromophores and enabled resolution of the emission of the 'red' subset of the protein's tryptophans. This avoided the non-radiative homo-energy transfer and enabled to study the fluorescence anisotropy decay kinetics of these residues without a distortion by the energy transfer (ET) process. Under these experimental conditions the fluorescence anisotropy decays practically from the limiting anisotropy value (0.3) for W in a bi-exponential process. The longer decay constant has a value larger than that expected for a global rotation of the HLA-A2/ peptide complex suggesting that the protein molecules exist in an oligomeric form. No clear assignment for the fast component can currently be made; it may be either a manifestation of a limited internal rotation of the W residues or a result of uncompleted compensation of the homo-ET process.
Bibliographical noteFunding Information:
We gratefully acknowledge support of the research reportedh ereb y the Minerva Foundation and the Crown Foundation for Immunological Research at The Weizmann Institute of Science and of NIH Grant CA47.554. The contribution of the Russell Memorial Foundation (Miami) helped in installation of the time-resolved equipment. The useful advice of Dr. David Garboczi and the measuremento f W distances by Mr. Michael Farazan are gratefully acknowledged.
- Anisotropy decay
- Fluorescence intensity
- Major histocompatibility complex, class I
- Non-radiative energy transfer
- Selective time-resolved fluorescence spectroscopy
- Tryptophan emission