Observing the osmophobic effect in action at the single molecule level

Abstract

Protecting osmolytes are widespread small organic molecules able to stabilize the folded state of most proteins against various denaturing stresses in vivo. The osmophobic model explains thermodynamically their action through a preferential exclusion of the osmolyte molecules from the protein surface, thus favoring the formation of intrapeptide hydrogen bonds. Few works addressed the influence of protecting osmolytes on the protein unfolding transition state and kinetics. Among those, previous single molecule force spectroscopy experiments evidenced a complexation of the protecting osmolyte molecules at the unfolding transition state of the protein, in apparent contradiction with the osmophobic nature of the protein backbone. We present single-molecule evidence that glycerol, which is a ubiquitous protecting osmolyte, stabilizes a globular protein against mechanical unfolding without binding into its unfolding transition state structure. We show experimentally that glycerol does not change the position of the unfolding transition state as projected onto the mechanical reaction coordinate. Moreover, we compute theoretically the projection of the unfolding transition state onto two other common reaction coordinates, that is, the number of native peptide bonds and the weighted number of native contacts. To that end, we augment an analytic Ising-like protein model with support for group-transfer free energies. Using this model, we find again that the position of the unfolding transition state does not change in the presence of glycerol, giving further support to the conclusions based on the single-molecule experiments.

Probing Small Molecule Binding to Unfolded Polyprotein Based on its Elasticity and Refolding

“…In our studies of urea and GuHCl in the submolar concentration range, we did not see a significant effect on binding to the unfolded polyprotein. This may indicate the binding is too weak at these concentrations, which is consistent with previous works showing that urea and GuHCl decrease the mechanical stability of the folded protein domains only at concentrations in the molar range (51)(52)(53)(54)(55).…”

Worm-Like Ising Model for Protein Mechanical Unfolding under the Effect of Osmolytes

“…Herein we show that the same lack of movement holds for various concentrations of DMSO and GndCl, indicating that osmolytes may not generally produce significant movements of the unfolding transition states of proteins. Furthermore, we expand the mentioned thermodynamic analysis with mechanical unfolding simulations based on the worm-like-chain (WLC) force-distance relation (12,13), showing that our Ising-like model exhibits no significant change in the unfolding distance of GB1 and I27 under the effect of various concentrations of DMSO, glycerol, and GndCl, in excellent agreement with the experimental results presented herein and those of Aioanei et al (9,10) and Cao and Li (11). Note that our Ising-like model lacks the expressive power to account for any possible structural role of osmolytes at the unfolding transition state of proteins, while still being able to explain the mentioned experimental data.…”

“…Theoretically, we have recently shown that an Ising-like model with support for the osmolyte effect does not exhibit any movement of the unfolding transition state of GB1 in the presence of glycerol 30% v/v, when projected thermodynamically onto a commonly used nonmechanical reaction coordinate (10). Herein we show that the same lack of movement holds for various concentrations of DMSO and GndCl, indicating that osmolytes may not generally produce significant movements of the unfolding transition states of proteins.…”

“…The osmolyte effect has been dissected into groupwise free energy contributions, with the protein backbone making up most of the protein's free energy of transfer to osmolytecontaining solutions (1). Making use of the group transfer free energies of amino-acid backbone units and side chains, a recent extension of the WSME model for the osmolyte effect enabled the thermodynamic projection of the protein energy landscape in the presence of osmolytes onto reaction coordinates commonly employed to monitor protein folding and unfolding (10).…”

Protein folding and stability in the presence of osmolytes

“…Reduction in osmolyte concentration in the immediate vicinity of the protein cannot be described solely based on the effect of the excluded volume, as in this case, the action of osmoprotectors on protein stability would be determined exclusively by the size of the osmolyte molecules [36]. An osmophobic character of the interactions of some osmolytes with a number of proteins has been shown experimentally [37,38]. It should be noted that introduction of osmolytes into solution under conditions of their exclusion from the immediate vicinity of the protein increases the chemical potential of the systems, i.e., it destabilizes it [25][26][27][28].…”