van der Waals interactions are important to protein stability and function. These interactions are usually identified empirically based on protein 3D structures. In this work, we performed a solution nuclear magnetic resonance (NMR) spectroscopy study of van der Waals interactions by detecting the through-space J-coupling between protein aliphatic side chain groups. Specifically, J-coupling values up to ∼0.5 Hz were obtained between the methyl and nearby aliphatic groups in protein GB3, providing direct experimental evidence for the van der Waals interactions. Quantum mechanical calculations suggest that the J-coupling is correlated with the exchange-repulsion term of van der Waals interaction. NMR detection of J-coupling offers a new tool to characterize such interactions in proteins.

A NMR protocol is introduced that permits accurate measurement of minute, remote chemical shift perturbations (CSPs), caused by a mutation-induced change in the electric field. Using protein GB3 as a model system, 1HN CSPs in K19A and K19E mutants can be fitted to small changes in the electric field at distal sites in the protein using the Buckingham equation, yielding an apparent dielectric constant εa of 8.6 ± 0.8 at 298 K. These CSPs, and their derived εa value, scale strongly with temperature. For example, CSPs at 313 K are about ∼30% smaller than those at 278 K, corresponding to an effective εa value of about 7.3 at 278 K and 10.5 at 313 K. Molecular dynamics simulations in explicit solvent indicate that solvent water makes a significant contribution to εa.

Residual dipolar couplings (RDCs) are commonly used in NMR for protein structure and dynamics studies, but it is challenging to generate five independent RDC data sets (required for simultaneous structure and dynamics determination) for most protein molecules in the magnetic field. In this work, a reporter protein with a lanthanide tag is introduced to create five independent alignments. This reporter protein is then attached to target proteins where five independent sets of RDCs are also obtained for the target proteins. The fitting of RDCs provides important information about the structure and dynamics of the target proteins. The method is simple and effective and, in principle, can be used to generate complete sets of RDCs for different protein molecules.

The intramolecular electric field (e-field) generated by protein GB3 side-chain charges K/E10, K/E19, and D/K40 was measured in the absence or presence of macromolecular crowding. The e-field responds differently to different crowding agents-dextran, Ficoll, BSA, and E. coli cell lysate. Dextran and Ficoll have no effect on the e-field. The lysate generally weakens the e-field but the amplitude of weakening varies greatly. For example, the e-field by K19 is reduced by 67% in the presence of 90 g/L lysate, corresponding to a charge change from 0.9 to 0.3 e for K19, whereas the e-fields by D/K40 are weakened only by ∼7% under the same lysate concentration. The extent of the e-field weakening by BSA is in between that by Ficoll (dextran) and lysate. Further investigations suggest that the e-field weakening mechanism by lysate is similar to that by NaCl. That is, the e-field generated by a protein surface charge affects the distribution of lysate which creates a reaction field and weakens the protein e-field. Our study indicates that the protein electrostatic property can be changed significantly due to quinary interaction with the cell environment.

Polyol and sugar osmolytes are commonly used in therapeutic protein formulations. How they may affect protein structure and function is an important question. In this work, through NMR measurements, we show that glycerol and sorbitol (polyols), as well as glucose (sugar), can shorten protein backbone hydrogen bonds. The hydrogen bond shortening is also captured by molecular dynamics simulations, which suggest a hydrogen bond competition mechanism. Specifically, osmolytes weaken hydrogen bonds between the protein and solvent to strengthen those within the protein. Although the hydrogen bond change is small, with the average experimental cross hydrogen bond 3hJNC′ coupling of two proteins GB3 and TTHA increased by ~ 0.01 Hz by the three osmolytes (160 g/L), its effect on protein function should not be overlooked. This is exemplified by the PDZ3−peptide binding where several intermolecular hydrogen bonds are formed and osmolytes shift the equilibrium towards the bound state.

Although numerous in vitro studies have shown that osmolytes are capable of stabilizing proteins, their effect on protein folding in vivo has been less understood. In this work, we investigated the effect of osmolytes, including glycerol, sorbitol, betaine, and taurine, on the folding of a protein GB3 variant in E. coli cells using NMR spectroscopy. 400 mM osmolytes were added to E. coli cells; only glycerol stabilizes the folded protein, whereas betaine and taurine considerably destabilize the protein through modulating folding and unfolding rates. Further investigation indicates that betaine and taurine can enhance the quinary interaction between the protein and cellular environment and manifestly weaken the electrostatic attraction in protein salt bridges. The combination of the two factors causes destabilization of the protein in E. coli cells. These factors counteract the preferential exclusion mechanism that is adopted by osmolytes to stabilize proteins.

Salt bridges are very common in proteins.But what drives the formation of protein salt bridges is not clear.Inthis work, we determined the strength of four salt bridges in the protein GB3 by measuring the DpK a values of the basic residues that constitute the salt bridges with ah ighly accurate NMR titration method at different temperatures.T he results show that the DpK a values increase with temperature,t hus indicating that the salt bridges are stronger at higher temperatures.Fitting of DpK a values to the vantHoff equation yields positive DHand DSvalues,thus indicating that entropydrives salt-bridge formation. Molecular dynamics simulations show that the protein and solvent make opposite contributions to DH and DS. Specifically,t he enthalpic gain contributed from the protein is more than offset by the enthalpic loss contributed from the solvent, whereas the entropic gain originates from the desolvation effect.

One important feature of hydrolysis of cellulose by cellulases is that the reaction slows down quickly after it starts. In this work, we investigate the slowdown mechanism at the early stage of the reaction using endoglucanase Tr. Cel5A-catalyzed phosphate acid-swollen cellulose (PASC) hydrolysis as a model system. Specifically, we focus on the effect of enzyme adsorption on the reaction slowdown. Nineteen single mutations are introduced (with the assistance of molecular dynamics simulations) to perturb the enzyme PASC interaction, yielding the adsorption partitioning coefficient Kr that ranged from 0.12 to 0.39 L/g, compared to that of the wild type (0.26 L/g). Several residues, including T18, K26, Y26, H229, and T300, are demonstrated to be important for adsorption of the enzyme to PASC. The kinetic measurements show that the slowdown of the hydrolysis is not correlated with the adsorption quantified by the partitioning coefficient Kr but is anticorrelated with the initial activity. This result suggests that the mutants with higher activity are more prone to being trapped or deplete the most reactive substrate faster and the adsorption plays no apparent role in the reaction slowdown. The initial activity of Cel5A against PASC is correlated with the enzyme specific activity against a soluble substrate p-nitrophenyl cellobioside.