Wavelength-resolved fluorescence emission of proteins using the synchrotron radiation as pulsed-light source: cross-correlations between lifetimes, rotational correlation times and tryptophan heterogeneity in FKBP59 immunophilin.
The time-resolved fluorescence intensity and anisotropy decays of the immunophilin domain of FKBP59 (FKBP59-I)--a protein containing two tryptophan residues (the W89, buried in a hydrophobic pocket and the W59, water exposed)--were studied using the time-correlated single photon counting (TCSPC) technique. The synchrotron radiation machine Super-ACO (Orsay, France) was used as a pulsed light source (approximately 8MHz). A mainly dual and discrete excited state lifetime distribution was previously evidenced (Rouvière et al., 1997). The lifetime heterogeneity has been suggested to be relevant to the topological tryptophan heterogeneity. Indeed, taking into account the spectroscopic properties of the single tryptophan residue of the immunophilin FKBP12, a highly homologous protein containing a single tryptophan residue, the short- and the long-lived lifetime species were assumed to be related to the solvent-buried and to the solvent-exposed fluorescent residues, respectively. We definitely demonstrate this point by describing the dynamical properties of each tryptophan residue of the FKBP59-I as a function of the emission wavelength. The data of the polarized components of the fluorescence emission were analyzed by the Maximum Entropy Method using a one-dimensional model (each excited-state lifetime tau being associated with each rotational correlation time theta) and a two-dimensional model (without any a priori association constraint between the tau's and the theta's). The two dimensional analysis of the polarized fluorescence intensity decays by MEM show the existence of a correlation between fast picosecond dynamics of the indole ring with the shortest-lived and blue emitting species. Conversely, the long-lived and red emitting population is mainly associated to the Brownian motion of the protein. A protein flexibility of the region located around the W59 residue, but slightly contributing to the light depolarization process, is also evidenced and can be specifically attributed to the red emitting population.
Vincent
M
M
Laboratoire pour l'Utilisation du Rayonnement Electromagnétique (LURE), Centre National de la Recherche Scientifique, Centre Universitaire Paris-Sud, Orsay, France.
Rouvière
N
N
Gallay
J
J
D005454:Fluorescence Polarization; D020104:Immunophilins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D017356:Synchrotrons; D022021:Tacrolimus Binding Proteins; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins