Selective steady-state and time-resolved fluorescence spectroscopy of an HLA-A2-peptide complex

• Published in: Immunology letters 1995-01, Vol.44 (2), p.195-201
• Main Authors: Gakamsky, Dmitry M., Haas, Elisha, Robbins, Penelope, Strominger, Jack L., Pecht, Israel
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anisotropy decay; Binding Sites; Fluorescence intensity; Fluorescence Polarization; HLA-A2 Antigen - chemistry; HLA-A2 Antigen - metabolism; Humans; Major histocompatibility complex, class I; Models, Molecular; Molecular Sequence Data; Non-radiative energy transfer; Peptides - chemistry; Peptides - metabolism; Protein Binding; Selective time-resolved fluorescence spectroscopy; Spectrometry, Fluorescence - methods; Time Factors; Tryptophan emission
• ISSN: 0165-2478; 1879-0542
• DOI: 10.1016/0165-2478(94)00214-C

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, 2 W β 2 m ) 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.