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

ORCiD

David M. Ojcius: 0000-0003-1461-4495

Department

Biomedical Sciences

Document Type

Article

Publication Title

Immunology Letters

ISSN

0165-2478

Volume

40

Issue

2

DOI

10.1016/0165-2478(94)90183-X

First Page

125

Last Page

132

Publication Date

5-1-1994

Abstract

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. Thus, differences between SC-Kd molecules binding low- and high-affinity peptides can be resolved by monitoring the emission properties of internal tryptophans. These results suggest a higher ‘compactness’ of the MHC molecules in the latter state, which can be rationalized in terms of an increase in number and strength of the bonding interactions that take place in the loaded SC-Kd complexes.

Link to Full Text

Share

COinS