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 comple xes of the single-chain H-2K(d) (SC-K-d) were studied by means of the single-photon counting technique. The latter enables time-resolved mea surements of fluorescence intensity and anisotropy decay parameters re levant to structural and dynamic properties of proteins. While the iso lated SC-K-d molecules in their 'original' purified form represent the unloaded state, i.e., containing endogenous low-affinity peptides, th e loaded SC-K-d protein is obtained by introducing well-defined high-a ffinity peptides that replace the low-affinity ones. These two SC-K-d forms were found to exhibit different time-resolved tryptophan emissio n patterns; the unloaded complexes show a slightly faster fluorescence intensity decay rate than the loaded one. Three well-resolved time do mains were distinguished in the anisotropy decay course of both forms: a short one in the picosecond range, an intermediate one of several n anoseconds, and a long one spanning several dozens to hundreds of nano seconds. They are assigned to superposition contributions of (short- a nd long-distance) non-radiative energy transfer processes, to motions of the tryptophans, and to rotation of the whole protein globule. In t he loaded SC-K(d)s, the first two processes were found to be attenuate d. It is therefore suggested that upon binding of high-affinity peptid es, the SC-K-d structure becomes more compact and certain tryptophans become less accessible to quenchers. The faster anisotropy decay obser ved in the unloaded form reflects both an enhancement in the energy-tr ansfer between the tryptophans and an acceleration of their motions. T hus, differences between SC-K-d molecules binding low- and high-affini ty peptides can be resolved by monitoring the emission properties of i nternal tryptophans. These results suggest a higher 'compactness' of t he MHC molecules in the latter state, which can be rationalized in ter ms of an increase in number and strength of the bonding interactions t hat take place in the loaded SC-K-d complexes.