Here we obtain the data needed to predict chemical interactions of polyethylene glycols (PEGs) and glycerol with proteins and related organic compounds, and thereby interpret or predict chemical effects of PEGs on protein processes. To accomplish this we determine interactions of glycerol and tetraEG with >30 model compounds displaying the major C, N, and O functional groups of proteins. Analysis of these data yields coefficients (α-values) quantifying interactions of glycerol, tetraEG and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with aromatic C, amide N, and cationic N, but unfavorably with amide O, carboxylate O and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliphatic C are quite favorable, while interactions of glycerol and PEG end groups with aliphatic C are not. Hence tetraEG and PEG 300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD) while glycerol, di- and mono-ethylene glycol are stabilizers. Favorable interactions with aromatic and aliphatic C explain why PEG400 greatly increases the solubility of aromatic hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α-values reported here, chemical contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA), and separated from excluded volume effects.

Quantitative information about amide interactions in water is needed to understand their contributions to protein folding and amide effects on aqueous processes, and to compare with computer simulations. Here we quantify interactions of urea, alkylated ureas and other amides by osmometry and amide-aromatic hydrocarbon interactions by solubility. Analysis of these data yields strengths of interaction of ureas and naphthalene with amide sp2O, amide sp2N, aliphatic sp3C, and amide and aromatic sp2C unified atoms in water. Interactions of amide sp2O with urea and naphthalene are favorable, while amide sp2O-alkyl urea interactions are unfavorable, becoming more unfavorable with increasing alkylation. Hence amide sp2O-amide sp2N interactions (proposed n–σ* hydrogen bond) and amide sp2O-aromatic sp2C (proposed n – π*) interactions are favorable in water, while amide sp2O-sp3C interactions are unfavorable. Interactions of all ureas with sp3C and amide sp2N are favorable and increase in strength with increasing alkylation, indicating favorable sp3C-amide sp2N and sp3C-sp3C interactions. Naphthalene results show that aromatic sp2C-amide sp2N interactions in water are unfavorable while sp2C-sp3C interactions are favorable. These results allow interactions of amide and hydrocarbon moieties and effects of urea and alkyl ureas on aqueous processes to be predicted or interpreted in terms of structural information. We predict strengths of favorable urea-benzene and N-methylacetamide interactions from experimental information to compare with simulations, and indicate how amounts of hydrocarbon and amide surface buried in protein folding and other biopolymer processes and transition states can be determined from analysis of urea and diethyl urea effects on equilibrium and rate constants.

Potassium glutamate (KGlu) is the primary Escherichia coli cytoplasmic salt. After sudden osmotic upshift, cytoplasmic KGlu concentration increases, initially because of water efflux and subsequently by K transport and Glu synthesis, allowing water uptake and resumption of growth at high osmolality. In vitro, KGlu ranks with Hofmeister salts KF and KSO in driving protein folding and assembly. Replacement of KCl by KGlu stabilizes protein-nucleic acid complexes. To interpret and predict KGlu effects on protein processes, preferential interactions of KGlu with 15 model compounds displaying six protein functional groups-sp (aliphatic) C; sp (aromatic, amide, carboxylate) C; amide and anionic (carboxylate) O; and amide and cationic N-were determined by osmometry or solubility assays. Analysis of these data yields interaction potentials (α-values) quantifying non-Coulombic chemical interactions of KGlu with unit area of these six groups. Interactions of KGlu with the 15 model compounds predicted from these six α-values agree well with experimental data. KGlu interactions with all carbon groups and with anionic (carboxylate) and amide oxygen are unfavorable, while KGlu interactions with cationic and amide nitrogen are favorable. These α-values, together with surface area information, provide quantitative predictions of why KGlu is an effective E. coli cytoplasmic osmolyte (because of the dominant effect of unfavorable interactions of KGlu with anionic and amide oxygens and hydrocarbon groups on the water-accessible surface of cytoplasmic biopolymers) and why KGlu is a strong stabilizer of folded proteins (because of the dominant effect of unfavorable interactions of KGlu with hydrocarbon groups and amide oxygens exposed in unfolding).

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Folding and other protein self-assembly processes are driven by favorable interactions between O, N, and C unified atoms of the polypeptide backbone and side chains. These processes are perturbed by solutes that interact with these atoms differently than water does. Amide NH···O=C hydrogen bonding and various π-system interactions have been better characterized structurally or by simulations than experimentally in water, and unfavorable interactions are relatively uncharacterized. To address this situation, we previously quantified interactions of alkyl ureas with amide and aromatic compounds, relative to interactions with water. Analysis yielded strengths of interaction of each alkylurea with unit areas of different hybridization states of unified O, N, and C atoms of amide and aromatic compounds. Here, by osmometry, we quantify interactions of 10 pairs of amides selected to complete this dataset. An analysis yields intrinsic strengths of six favorable and four unfavorable atom−atom interactions, expressed per unit area of each atom and relative to interactions with water. The most favorable interactions are sp2O−sp2C (lone pair−π, presumably n−π*), sp2C−sp2C (π−π and/or hydrophobic), sp2O−sp2N (hydrogen bonding) and sp3C−sp2C (CH−π and/or hydrophobic). Interactions of sp3C with itself (hydrophobic) and with sp2N are modestly favorable, while sp2N interactions with sp2N and with amide/aromatic sp2C are modestly unfavorable. Amide sp2O−sp2O interactions and sp2O−sp3C interactions are more unfavorable, indicating the preference of amide sp2O to interact with water. These intrinsic interaction strengths are used to predict interactions of amides with proteins and chemical effects of amides (including urea, N-ethylpyrrolidone [NEP], and polyvinylpyrrolidone [PVP]) on protein stability.