SELECTIVE STEADY-STATE AND TIME-RESOLVED FLUORESCENCE SPECTROSCOPY OFAN HLA-A2-PEPTIDE COMPLEX

The human class I major histocompatibility complex (MHC) encoded molec ule HLA-A2 loaded with the high-affinity peptide GILGRVFTL (p790), was studied by means of steady-state and picosecond fluorescence intensit y and fluorescence anisotropy methods. The large number of tryptophan residues (W) (10 W/heavy chain, 2 W/beta(2)m) as well as their fluores cence sensitivity to the microenvironment, determine the emission of t he studied complex. The HLA-A2/peptide complex exhibits a considerable static inhomogeneous broadening (IB) of the W electronic spectra, whi ch results in a dependence of the steady-state fluorescence spectrum o n the excitation wavelength. The high concentration of W's chromophore s and the spectral IB cause a directed non-radiative migration of elec tronic excitation energy by Foerster's mechanism from 'blue' W residue s to 'red' ones. This phenomenon manifests itself in a nanosecond fluo rescence 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 resolutio n of the emission of the 'red' subset of the protein's tryptophans. Th is avoided the non-radiative home-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 experime ntal conditions the fluorescence anisotropy decays practically from th e limiting anisotropy value (0.3) for W in a bi-exponential process. T he longer decay constant has a value larger than that expected for a g lobal rotation of the HLA-A2/peptide complex suggesting that the prote in 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 uncom pleted compensation of the homo-ET process.