Picosecond fluorescence spectroscopy of a single-chain class I major histocompatibility complex encoded protein in its peptide loaded and unloaded states.

The tryptophan fluorescence properties of two different peptide complexes of the single-chain H-2Kd (SC-Kd) were studied by means of the single-photon counting technique. The latter enables time-resolved measurements of fluorescence intensity and anisotropy decay parameters relevant to structural and dynamic properties of proteins. While the isolated SC-Kd molecules in their 'original' purified form represent the unloaded state, i.e., containing endogenous low-affinity peptides, the loaded SC-Kd protein is obtained by introducing well-defined high-affinity peptides that replace the low-affinity ones. These two SC-Kd forms were found to exhibit different time-resolved tryptophan emission patterns; the unloaded complexes show a slightly faster fluorescence intensity decay rate than the loaded one. Three well-resolved time domains were distinguished in the anisotropy decay course of both forms: a short one in the picosecond range, an intermediate one of several nanoseconds, and a long one spanning several dozens to hundreds of nanoseconds. They are assigned to superposition contributions of (short- and long-distance) non-radiative energy transfer processes, to motions of the tryptophans, and to rotation of the whole protein globule. In the loaded SC-Kds, the first two processes were found to be attenuated. It is therefore suggested that upon binding of high-affinity peptides, the SC-Kd structure becomes more compact and certain tryptophans become less accessible to quenchers. The faster anisotropy decay observed in the unloaded form reflects both an enhancement in the energy-transfer between the tryptophans and an acceleration of their motions.(ABSTRACT TRUNCATED AT 250 WORDS)