Continuous dissolution of structure during the unfolding of a small protein

Abstract

The unfolding kinetics of many small proteins appears to be first order, when measured by ensemble-averaging probes such as fluorescence and circular dichroism. For one such protein, monellin, it is shown here that hidden behind this deceptive simplicity is a complexity that becomes evident with the use of experimental probes that are able to discriminate between different conformations in an ensemble of structures. In this study, the unfolding of monellin has been probed by measurement of the changes in the distributions of 4 different intramolecular distances, using a multisite, time-resolved fluorescence resonance energy transfer methodology. During the course of unfolding, the protein molecules are seen to undergo slow and continuous, diffusive swelling. The swelling process can be modeled as the slow diffusive swelling of a Rouse-like chain with some additional noncovalent, intramolecular interactions. Here, we show that specific structure is lost during the swelling process gradually, and not in an all-or-none manner, during unfolding.diffusive swelling ͉ distance distribution ͉ gradual unfolding ͉ Rouse model ͉ time-resolved FRET

Gradual Folding of an Off-Pathway Molten Globule Detected at the Single-Molecule Level

“…At equilibrium, several of these on-pathway intermediates were shown to form in a gradual manner depending on GuHCl, urea or Na2SO4 concentration or pH and temperature. Continuous, diffusive swelling was observed also in a time-resolved study of the unfolding of folding intermediates of monellin [60]. In contrast, hopping observed in a force spectroscopy experiment of ribonuclease H folding shows that a single activation barrier separates unfolded molecules from a MG intermediate, and that the corresponding transition is first order [61].…”

Kinetic Studies of the Folding of Heterodimeric Monellin: Evidence for Switching between Alternative Parallel Pathways

“…7,10,42 The study of the unfolding of scMN has shown that unfolding commences through the formation of a dry molten globule 43 and proceeds gradually and not in an all-or-none manner. 44 Until recently, there were only a few quantitative studies of the unfolding of dcMN at equilibrium, 37,45 but the recent development of a good expression system for dcMN 46 made it possible to make a detailed study of its unfolding at equilibrium in relation to that of scMN. 39 This has set the stage for quantitative studies of its folding mechanism.…”

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

“…However, Kayser et al found it to be a particularly useful tool for studying structure-function relationships in monoclonal antibodies, which were found to have an unusually high Trp content [199]. Another use of Trp as a natural fluorophore for FRET is to probe protein folding/unfolding, an important yet poorly understood biological process [200][201][202]. For example, Jha et al used FRET to study the unfolding of a small protein, monellin [202].…”

“…Another use of Trp as a natural fluorophore for FRET is to probe protein folding/unfolding, an important yet poorly understood biological process [200][201][202]. For example, Jha et al used FRET to study the unfolding of a small protein, monellin [202]. The transition of this small protein, from an unfolded to a folded state, is not completely understood but by using a naturally occurring Trp and additionally labeling the protein with the FRET acceptor thionitrobenzoate, researchers were able to determine that monellin unfolds gradually rather than all in one motion [202].…”

“…For example, Jha et al used FRET to study the unfolding of a small protein, monellin [202]. The transition of this small protein, from an unfolded to a folded state, is not completely understood but by using a naturally occurring Trp and additionally labeling the protein with the FRET acceptor thionitrobenzoate, researchers were able to determine that monellin unfolds gradually rather than all in one motion [202]. Visser et al utilized Trp homo-FRET to characterize protein folding of apoflavodoxin, demonstrating the potential of this method as a powerful means to understand some of the still unknown mechanisms of protein folding [201].…”