Fluorescence and molecular dynamics study of the internal motion of the buried tryptophan in bacteriophage T4 lysozyme: Effects of temperature and alteration of nonbonded networks

Abstract

Results from time-resolved polarization studies of tryptophan emission in bacteriophage T4 lysozyme variants are compared with results from molecular dynamics calculations. Wild-type bacteriophage T4 lysozyme has three tryptophans in environments differing in nonbonded contacts and solvent exposure. The emission spectra of these three tryptophan residues are identical but their internal dynamics are quite distinct. This difference in dynamics is in agreement with the reorientational correlation functions calculated from molecular dynamics trajectories. The most buried tryptophan (W 138) is immobile at 20 °C in the wild-type enzyme but undergoes internal libration at elevated temperatures. Amino acid substitutions at sequence positions 105 and 146 that alter the nonbonded contacts of this tryptophan result in large amplitude internal motion and redshifted fluorescence emission spectra. The magnitudes of the Stokes shifts for the emission of W138 are well correlated with the rates of trypsin proteolysis of these lysozymes. Addition of a disulfide bridge local to these substitution sites reverses the effects of the substitutions on the tryptophan dynamics and emission spectra. The data suggest that fluctuations responsible for “gating” the internal motion of this buried tryptophan in the native state of the protein also result in transient exposure to solvent. Analyses of molecular dynamics trajectories of lysozyme variants with amino acid substitutions show that perturbations of nonbonded networks found in the wild-type enzyme result in increased local fluctuations, reorientation and solvent exposure of buried residues.