Identification of Multiple Folding Pathways of Monellin Using Pulsed Thiol Labeling and Mass Spectrometry

Abstract

Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.

Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride

“…MMTS-labeling experiments were performed as described previously (33,35). In brief, RNase H was unfolded in 3.4 M GdmCl inside a quenched-flow machine (SFM-400 Q/S from Biologic), and a 6-ms pulse of 20 mM MMTS was applied at different times of unfolding.…”

“…1A). We used two different thiol-specific labeling reagents, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and methyl metahnethiosulfonate (MMTS), both of which react rapidly with exposed thiolate groups (33)(34)(35), to measure the changes in solvent accessibility during unfolding ( Fig. 5 A−C).…”

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

“…Nevertheless, and not surprisingly perhaps, it has been shown that the fast phase seen in the folding of scMN also leads to the formation of two intermediates of differing stabilities on two competing pathways. 7,42 Kinetic model for the folding of dcMN A global fitting program, written in MATLAB, was used to quantitatively analyze the dependence of the refolding kinetics on GdnHCl concentration, according to Scheme 3. Details about the fitting program are provided in Materials and Methods.…”

“…2,3 Multiple pathways have indeed been observed to operate during the folding and unfolding of several proteins. [4][5][6][7][8][9][10][11][12] Much is yet to be learnt about how competing folding pathways differ in the sequence of structural events defined by each pathway, as well as about the nature of the free energy barriers on a folding pathway. 13,14 Multiple folding pathways are associated with structural heterogeneity in the unfolded state [15][16][17][18] and intermediates.…”

“…38,39 The study of the folding of scMN has yielded much useful insights about chain collapse 40,41 and the utilization of multiple pathways during folding. 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.…”