Osmolytes are essential for cellular function under ubiquitous osmotic stress. Trimethylamine N-oxide (TMAO) is one such osmolyte that has gained remarkable attention due to its protein-protective ability against urea. This Review aims at providing a detailed account of recent theoretical and experimental developments in characterizing the structural changes and thermodynamic stability of proteins in the presence of TMAO and urea. New vapor pressure osmometry and molecular dynamics simulation results on urea–TMAO solutions are presented, and a unified molecular mechanism of TMAO counteraction of urea-induced protein denaturation is introduced. In addition, a detailed technical assessment of molecular dynamics force fields for TMAO and for urea–TMAO solutions is presented. The force field analysis highlights how many of the commonly used force field models are in fact incompatible with solvation thermodynamics and can lead to misleading conclusions. A new optimized force field for TMAO (Shea(m)) is presented, and a recently optimized force field for TMAO–urea (Netz(m)) that best reproduces experimental data is highlighted.
The osmolyte trimethylamine-N-oxide (TMAO) is able to increase the thermodynamic stability of folded proteins, counteracting pressure denaturation. Herein, we report experimental solubility data on penta-alanine (pAla) in aqueous TMAO solutions (at pH = 7 and pH = 13) together with molecular simulation data for pAla, penta-serine (pSer), and an elastin-like peptide (ELP) sequence (VPGVG) under varying pH and pressure conditions. The effect of the peptide end groups on TMAO− peptide interactions is investigated by comparing the solvation of zwitterionic and negatively charged pentamers with the solvation of pentamers with charge-neutral C-and N-termini and linear, virtually infinite, peptide chains stretched across the periodic boundaries of the simulation cell. The experiments and simulations consistently show that TMAO is net-depleted from the pAla−water interface, but local accumulation of TMAO is observed just outside the first hydration shell of the peptide. While the same observations are also made in the simulations of the zwitterionic pentamers (Ala, Ser, and ELP) and virtually infinite peptide chains (Ala and ELP), weak preferential binding of TMAO is instead observed for pAla with neutral end groups at a 1 M TMAO concentration and for an ELP pentamer with capped neutral end groups at a 0.55 M TMAO concentration studied in previous work (Y.-T. Liao et al. Proc. Natl. Acad. Sci. USA, 2017USA, , 114, 2479USA, −2484. The above observations made at 1 bar ambient pressure remain qualitatively unchanged at 500 bar and 2 kbar. Local accumulation of TMAO correlates with a reduction in the total number of peptide−solvent hydrogen bonds, independent of the peptide's primary sequence and the applied pressure. By weakening water hydrogen bonds with the protein backbone, TMAO indirectly contributes to stabilizing internal hydrogen bonds in proteins, thus providing a protein stabilization mechanism beyond net depletion.
The behavior of thermoresponsive polymer poly(Nisopropylacrylamide) (PNiPAM), an essential building block in the design of smart soft materials, in aqueous solutions has attracted much interest, which contrasts with our knowledge of Nisopropylacrylamide (NiPAM) monomer. Strikingly, the physicochemical properties of aqueous NiPAM are similarly rich, and their understanding is far from being complete. This stems from the lack of accurate thermodynamic data and quantitative model for atomistic simulations. In this joint study, we have probed the thermodynamic behavior of aqueous NiPAM by experimental methods, molecular dynamics (MD) simulations, and Kirkwood− Buff (KB) analysis at ambient conditions. From the partial molar volumes and simultaneously correlated osmotic coefficients, with excess partial molar enthalpies of NiPAM in water, the concentration and temperature dependence of KB integrals was determined. For the purpose of this work, we have developed and employed a novel NiPAM force field, which not only reproduces KB integrals (G ij ) and adequately captures macroscopic thermodynamic quantities but also provides more accurate structural insight than the original force fields. We revealed in the vicinity of NiPAM the competing effect of amide hydration with interaction between nonpolar regions. This microscopic picture is reflected in the experimentally observed NiPAM−NiPAM association, which is present from highly dilute conditions up to the solubility limit and is evidenced by G 22 . From intermediate concentrations, it is accompanied by the existence of apparent dense-water regions, as indicated by positive G 11 values. The here-employed KB-based framework provided a mutually consistent thermodynamic and microscopic insight into the NiPAM solution and may be further extended for ion-specific effects. Moreover, our findings contribute to the understanding of thermodynamic grounds behind PNiPAM collapse transition.
Using a combination of molecular dynamics simulation, dialysis experiments, and electronic circular dichroism measurements, we studied the solvation thermodynamics of proteins in two osmolyte solutions, trimethylamine N-oxide (TMAO) and betaine. We showed that existing force fields are unable to capture the solvation properties of the proteins lysozyme and ribonuclease T1 and that the inaccurate parametrization of protein–osmolyte interactions in these force fields promoted an unphysical strong thermal denaturation of the trpcage protein. We developed a novel force field for betaine (the KBB force field) which reproduces the experimental solution Kirkwood–Buff integrals and density. We further introduced appropriate scaling to protein–osmolyte interactions in both the betaine and TMAO force fields which led to successful reproduction of experimental protein–osmolyte preferential binding coefficients for lysozyme and ribonuclease T1 and prevention of the unphysical denaturation of trpcage in osmolyte solutions. Correct parametrization of protein–TMAO interactions also led to the stabilization of the collapsed conformations of a disordered elastin-like peptide, while the uncorrected parameters destabilized the collapsed structures. Our results establish that the thermodynamic stability of proteins in both betaine and TMAO solutions is governed by osmolyte exclusion from proteins.
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