Continuous Dissolution Of Structure During The Unfolding Of A Small Protein

Abstract

An unresolved question in the field of protein folding is whether a protein unfolds in a two-state (N↔U) cooperative manner with only two species being populated during the entire unfolding reaction or in a non-cooperative fashion with a continuum of intermediate forms being populated. To make a definitive distinction between the two has been a difficult challenge, because of the difficulty in identifying and quantifying populations of different species present together during the unfolding reaction. Time-resolved fluorescence resonance energy transfer (TR-FRET) method can differentiate and measure selectively the populations of N, U and I forms, if present together. In this method, energy transfer efficiency is estimated by collecting the decays of fluorescence intensity of the donor fluorophore in the presence or absence of an acceptor. When such fluorescence intensity decays are analyzed by the maximum entropy method (MEM), distributions of fluorescence lifetimes are obtained, which can be used to generate a distribution of distances between the donor and acceptor. In this study, a multi-site, TR-FRET methodology coupled to MEM analysis has been used, for the first time, to study the time evolution of the probability distributions of four intra-molecular distances in the small plant protein monellin, as it unfolds starting from the native state. Surprisingly, one distance is seen to increase completely in a gradual manner, while the increase in the other three distances appears to have both discrete and gradual components. Hence, the protein is found to sample many intermediate conformations, characterized by different intra-molecular distances, before unfolding completely. The observed data can be explained by a simple physical model based upon swelling of a homopolymer chain undergoing diffusive dynamics according to the Rouse model.