Forcing the reversibility of a mechanochemical reaction

Abstract

Mechanical force modifies the free-energy surface of chemical reactions, often enabling thermodynamically unfavoured reaction pathways. Most of our molecular understanding of force-induced reactivity is restricted to the irreversible homolytic scission of covalent bonds and ring-opening in polymer mechanophores. Whether mechanical force can by-pass thermodynamically locked reactivity in heterolytic bimolecular reactions and how this impacts the reaction reversibility remains poorly understood. Using single-molecule force-clamp spectroscopy, here we show that mechanical force promotes the thermodynamically disfavored S_N2 cleavage of an individual protein disulfide bond by poor nucleophilic organic thiols. Upon force removal, the transition from the resulting high-energy unstable mixed disulfide product back to the initial, low-energy disulfide bond reactant becomes suddenly spontaneous, rendering the reaction fully reversible. By rationally varying the nucleophilicity of a series of small thiols, we demonstrate how force-regulated chemical kinetics can be finely coupled with thermodynamics to predict and modulate the reversibility of bimolecular mechanochemical reactions.

Fig. 1

Thermodynamically unfavoured disulfide bond reduction. a The distinct physicochemical and reactivity properties of a number of small thiol-rich nucleophiles are exemplified in their largely different sulfur pK_a, spanning the range pKa ~7–10 (individual values represented on top of the chemical structures). b DFT calculations of the standard free energy (M06-2X functional with one explicit water) associated with disulfide rupture of cystine by the different thiol nucleophiles are in good agreement with the experimentally measured pK_a values. c The Ellman’s assay measures the reactivity of the small low molecular weight thiols in solution, displaying a reactivity trend that increases with the sulfur pK_a. d Mass spectrometry experiments measure the ability of each nucleophilic thiol to reduce an individual disulfide bond embedded within the core of a I27_E24C-K55C protein (inset) under 1:54 (protein disulfide:active nucleophile) conditions upon acetonitrile exposure (green, thermodynamically allowed; orange, marginally allowed). The % of reduced disulfides matches the reactivity trend observed in solution and predicted by DFT calculations. (Empty circles represent values from individual experiments, n = 3, error bars: s.d)

Fig. 2

Mechanical force can induce thermodynamically non-spontaneous disulfide bond cleavage. a Schematic of the single-molecule force-clamp experiment, whereby a single (I27_E24C-K55C)₈ polyprotein is stretched by an AFM cantilever. b In the mechanochemical assay, mechanical force unfolds the protein (gray region) up to the mechanically rigid disulfide bond (formed between cysteine 24 and 55), which becomes solvent exposed. The presence of a nucleophile triggers disulfide cleavage, fingerprinted by the 10 nm steps that correspond to the extension of the protein amino acids that were trapped behind the disulfide bond (red region). The resulting reduced and mechanically unfolded protein harbors a mixed disulfide and a reduced protein thiolate. c Individual reduction trajectory demonstrating that the protein disulfide bond can be cleaved by 2 mM cysteine-methyl-ester (black asterisks mark the reduction of each individual disulfide bond under force, fingerprinted by a stepwise increase in the protein length of ~10 nm). d Disulfide bond rupture kinetics at F = 350 pN for the thermodynamically largely disfavored disulfide bond reduction by Cys-ME (red) and the marginally un-favored reaction in the presence of penicillamine (dark orange), compared to that of L-cysteine (orange). All compounds were kept at a constant deprotonated concentration [S⁻] = 2 mM. e The concentration-normalized rate of rupture for all compounds at a constant F = 350 pN correlates with the electrostatic partial charge localized on the nucleophilic sulfur. (Inset: charge distribution plot of Cys-ME, from red (negative), to green (~0) to blue (positive)) (From left to right n = 13, 24, 23, 17, 35, 23, 34, and 17, where n = number of individual trajectories). f The force-dependent reduction kinetics for NAC, thioglycerol, mesna, and glutathione demonstrates that all compounds exhibit a similar force-sensitivity yet different nucleophilicity; (From left to right: n_NAC = 35, 17, 42, and 24; n_Thioglycerol = 19, 35, 21, and 27; n_Mesna = 19, 35, 21, and 27; n_Glutathione = 37, 17, 41, and 16; error bars: s.d)

Fig. 3

Mechanical activation of thermodynamically unfavoured reactions induces chemical reversibility. a Disulfide bond reduction by mesna (ΔG° = −6.7 kcal mol⁻¹, green) results in a low-energy mixed disulfide that cannot reversibly reform the initial disulfide bond in the absence of force, marked by the absence of ~10 nm steps in the test pulse. b Conversely, reduction by Cys-ME (ΔG° = + 7.3 kcal mol⁻¹, red) gives rise to subsequent disulfide bond reformation, hallmarked by the ~10 nm stepwise increase in the test pulse. c The extent of disulfide bond reformation for all the tested compounds (green, thermodynamically allowed; orange, marginally allowed and red, thermodynamically unfavoured) exhibits a linear correlation with the pK_a (r² = 0.88); (From left to right n = 16, 35, 30, 24, 32, 33, 53, 31, and 39; error bars: s.d)

Fig. 4

Mechanical force deforms the reaction energy landscape and modulates the reversibility of the reaction. Schematics of the 1D energy landscapes highlighting how the application of mechanical force to an individual protein disulfide in the presence of chemically distinct small thiolates induces disulfide bond rupture and governs the reaction reversibility. Using a thiol nucleophile that renders the disulfide bond cleavage reaction thermodynamically spontaneous (ΔG < 0), mechanical force will lower the main energy barrier (dashed arrow), giving rise to a low free energy mixed-disulfide. Consequently, the reverse reaction (entailing the S_N2 reattack of the mixed disulfide bond by the neighboring protein reduced thiolate) will not be favorable, making the overall net reaction irreversible (red solid arrow). By contrast, mechanical force can trigger a thermodynamically un-favored reaction (ΔG > 0) by also significantly lowering its activation energy (dashed arrow). In this case, the resulting mixed disulfide bond has higher energy than the initial reactants. Hence, upon force release, the reformation of the initial disulfide bond becomes suddenly spontaneous, rendering the overall reaction reversible (green solid arrow)