Effect of unfolding on the tryptophanyl fluorescence lifetime distribution in apomyoglobin

E Bismuto,E Gratton,G Irace

doi:10.1021/bi00406a047

Abstract

Proteins exhibit, even in their native state, a large number of conformations differing in small details (substates). The fluorescence lifetime of tryptophanyl residues can reflect the microenvironmental characteristics of these subconformations. We have analyzed the lifetime distribution of the unique indole residue of tuna apomyoglobin (Trp A-12) during the unfolding induced by temperature or guanidine hydrochloride. The results show that the increase of the temperature from 10 to 30 degrees C causes a sharpening of the lifetime distribution. This is mainly due to the higher rate of interconversion among the conformational substates in the native state. A further temperature increase produces partially or fully unfolded states, resulting in a broadening of the tryptophanyl lifetime distribution. The data relative to the guanidine-induced unfolding show a sigmoidal increase of the distribution width, which is due to the transition of the protein structure from the native to the random-coiled state. The broadening of the lifetime distribution indicates that, even in the fully unfolded protein, the lifetime of the tryptophanyl residues is influenced by the protein matrix, which generates very heterogeneous microenvironments.

Highlights

$University of Illinois at Urbana-Champaign, 0006-2960/88/0427-2132$01.50/0 transition is completed, proteins are found to be random coiled, without any residual ordered structure (Tanford, 1968)
The results indicate that the protein unfolding is associated with a progressive broadening of the tryptophanyl lifetime distribution which can be related to a larger number of conformational substates in the unfolded state
The emission spectrum at 20 "C of tuna apomyoglobin excited at 298 nm shows, in the absence of denaturant, an emission maximum centered at 321 nm

Summary

Introduction

$University of Illinois at Urbana-Champaign, 0006-2960/88/0427-2132$01.50/0 transition is completed, proteins are found to be random coiled, without any residual ordered structure (Tanford, 1968). In these conditions, the spectroscopic properties of the aromatic residues are supposed to be similar to those of the monomeric amino acids in water (Demchenko, 1986). Static as well as dynamic considerations justify the use of continuous distributions in the analysis of the fluorescence decay rates of proteins (Alcala et al, 1987a,b). The results indicate that the protein unfolding is associated with a progressive broadening of the tryptophanyl lifetime distribution which can be related to a larger number of conformational substates in the unfolded state

Methods

Results

Conclusion