A Nanosecond Time-Resolved Fluorescence Study of Recombinant Human Myelin Basic Protein

Abstract

Myelin basic protein (MBP) is a major component of myelin and plays a central role in the maintenance of its compact multilayered structure. Tryptophan fluorescence is sensitive to environmental factors such as polarity and rotational mobility and often reflects conformational changes in proteins. This work describes a detailed examination of the time-resolved emission properties of the single tryptophan of recombinant human myelin basic protein. Fluorescence decay curves were collected at two separate excitation wavelengths and at different wavelengths across the emission spectrum. The fitting parameters obtained with the aid of global analysis were combined with steady-state emission spectra collected from the same samples and are presented as decay-associated spectra (DAS) and time-resolved emission spectra (TRES). The effects of temperature and binding to anionic membranes on the decay of emission of MBP were investigated. The changes in fitted parameters and the appearance of DAS and TRES as a function of experimental conditions are interpreted in terms of variation in the local environment of the single tryptophan in MBP. The implications of the changes in local environment resulting from experimental treatments are discussed in the context of the overall conformation of MBP and are compared to structural and photophysical properties of MBP obtained from the central nervous system tissue of several species [P. Cavatorta, S. Giovanelli, A. Bobba, P. Riccio, and E. Quagliariello, Acta Neurol. (Napoli) 13, 162–169, (1991; P. Cavatorta, S. Giovanelli, A. Bobba, P. Riccio, A. G. Szabo, and E. Quagliarello, Biophys. J. 66, 1174–1179, 1994; P. Cavatorta, L. Masotti, A. G. Szabo, D. Juretic, P. Riccio, and E. Quagliariello, Cell Biophys. 13, 201–215, 1988].