Characterization of the Folding and Unfolding Reactions of Single-Chain Monellin:  Evidence for Multiple Intermediates and Competing Pathways

Abstract

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by...

Continuous dissolution of structure during the unfolding of a small protein

“…This study has further brought out the complexity of the unfolding reaction by showing that the initially formed intermediate ensembles, I 1 and I 2 , unfold gradually, and not just in a few steps as indicated in the study in ref. 27. Here, it is shown that I 1 and I 2 possess native-like structure, as judged by the observation of only a very marginal loss in secondary structure at 150 ms of unfolding (Fig.…”

“…In addition to WT MNEI (Cys42), 3 more mutant variants, C42AS68C (Cys68), C42AT82C (Cys82) and C42AP97C (Cys97), each with a single tryptophan residue (Trp4) and a single cysteine residue were generated by site-directed mutagenesis, and the proteins purified as described in ref. 27. The TNB-labeled proteins were obtained as described in SI Appendix.…”

“…It is likely that I 1 and I 2 represent the products of 2 parallel unfolding reactions, in which many intrachain noncovalent interactions break in different parts of the chain. In fact, in an earlier study of the unfolding of MNEI (27), native protein had been observed to transform into differentially unfolded forms (15% and 85%) within a few ms of unfolding. Here, it appears that I 1 and I 2 form with probability of 0.2 and 0.8, respectively (Figs.…”

“…Earlier studies of the folding and unfolding of monellin (26)(27)(28) had shown that both processes are complex and mediated by multiple compact intermediates. This study has further brought out the complexity of the unfolding reaction by showing that the initially formed intermediate ensembles, I 1 and I 2 , unfold gradually, and not just in a few steps as indicated in the study in ref.…”

“…Single chain monellin (MNEI) is a sweet plant protein, whose folding and unfolding reactions have been studied extensively (26)(27)(28). Here, 4 single cysteine, single tryptophan containing mutant forms of MNEI have been used (Fig.…”