For proteins that denature irreversibly, the denaturation is typically triggered by a partial unfolding, followed by a permanent change (e.g., aggregation). The regions that initiate the partial unfolding are named “weak spots”. In this work, a molecular dynamics (MD) simulation and data analysis protocol is developed to identify the weak spots of Trichoderma reesei Cel7B, an important endoglucanase in cellulose hydrolysis, through assigning the local melting temperature (Tmp) to individual residue pairs. To test the predicted weak spots, a total of eight disulfide bonds were designed in these regions and all enhanced the enzyme thermostability. The increased stability, quantified by ΔT50 (which is the T50 difference between the mutant and the wild type enzyme), is negatively correlated with the MD-predicted Tmp, demonstrating the effectiveness of the protocol and highlighting the importance of the weak spots. Strengthening interactions in these regions proves to be a useful strategy in improving the thermostability of Tr. Cel7B.

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.

AbstractSalt bridges are very common in proteins. But what drives the formation of protein salt bridges is not clear. In this work, we determined the strength of four salt bridges in the protein GB3 by measuring the ΔpKa values of the basic residues that constitute the salt bridges with a highly accurate NMR titration method at different temperatures. The results show that the ΔpKa values increase with temperature, thus indicating that the salt bridges are stronger at higher temperatures. Fitting of ΔpKa values to the van't Hoff equation yields positive ΔH and ΔS values, thus indicating that entropy drives salt-bridge formation. Molecular dynamics simulations show that the protein and solvent make opposite contributions to ΔH and ΔS. Specifically, the 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.

The presence and extent of hydrogen-bonding (H-bonding) cooperativity in proteins remains a fundamental question, which in the past has been studied extensively, mostly by infrared and fluorescence measurements on model peptides. We demonstrate that such cooperativity can be studied in an intact protein by hydrogen/deuterium exchange NMR spectroscopy. The method is based on the fact that substitution of NH by ND in a backbone amide group slightly weakens the N–H···O═C hydrogen bond. Our results show that such substitution at position i in an α-helix impacts the 1H and 15N chemical shifts of the amide sites of residues i – 3 to i + 3. Quantum mechanical calculations indicate that the upfield shifts of 1H and 15N resonances at site i, observed upon H/D exchanges at sites i – 3, i + 1, i + 2, and i + 3, correspond to a decrease of the ith backbone amide electric dipole moment, which weakens its H-bonding and long-range electrostatic interactions with other backbone amides in the α-helix. These results provide new quantitative insights into the cooperativity of H-bonding in protein α-helices.

•The electron transfer isomer observed in anionic triads is useful for mechanism study of biology process and bioactivity.•The difference between two hydrogen bonds distance is enlarged by solvent dielectric constant.•Distinct variation of the IR spectra characterized the electron transfer isomers.•The variations of chemical shift and spin-spin coupling constant are more significant upon electron transfer than solvent dielectric constant.•The augmentation of solvent dielectric constant stabilizes the system and enhances the difference of isomers.The hydrogen bond plays a vital role in structural arrangement, intermediate state stabilization, materials function, and biological activity of certain enzymatic reactions. The solvent and electronic effects on hydrogen bonds are illustrated employing the polarizable contimuum model at B3LYP/6–311++G(d,p) level. Geometry optimizations reflect the significant solvent and electronic effect. The proton departs spontaneously upon oxidation from the hydroxyl group of tyrosyl in hydrogen bonded Tyr⋯Asp⋯Arg triads in both gas phase and solvents. The electron transfer isomers are observed for anionic triads, no matter what the solvent is. The difference of distance between two hydrogen bonds is enlarged in solvent as compared to that in gas phase. The electronic effect on IR spectra is distinctive. The tyrosyl fragment tends to be oxidized and the arginine moiety is easier to bind an excess electron. The variations of chemical shift and spin-spin coupling constant are more significant upon electron transfer than upon solvent dielectric constant. The augmentation of solvent dielectric constant stabilizes the system, enhances the difference of isomers, and increases the vertical ionization potential and vertical electron affinity values.Hydrogen bond plays vital role in structural arrangement, material function, and biological activity of certain enzymatic reactions. The generation or broken of a hydrogen bond varies the kinetics or mechanism of an enzyme reaction. Solvent effect on hydrogen bonded Tyr⋯Asp⋯Arg triads are explored from the geometrical, NBO charges, IR spectra, NMR parameters, and energy viewpoints. The detection and characterization of electron transfer isomer offer useful informaion for mechanism study of biology process and bioactivity.

Hydrogen bonds or salt bridges are usually formed to stabilize the buried ionizable residues. However, such interactions do not exist for two buried residues D271 and E305 of Trichoderma reesei Cel5A, an endoglucanase. Mutating D271 to alanine or leucine improves the enzyme thermostability quantified by the temperature T50 due to the elimination of the desolvation penalty of the aspartic acid. However, the same mutations for E305 decrease the enzyme thermostability. Free energy calculations based on the molecular dynamics simulation predict the thermostability of D271A, D271L, and E305A (compared to WT) in line with the experimental observation but overestimate the thermostability of E305L. Quantum mechanical calculations suggest that the carboxyl–peptide plane stacking interactions occurring to E305 but not D271 are important for the carboxyl group stabilization. For the protonated carboxyl group, the interaction energy can be as much as about −4 kcal/mol for parallel stacking and about −7 kcal/mol for T-shaped stacking. For the deprotonated carboxyl group, the largest interaction energies for parallel stacking and T-shaped stacking are comparable, about −7 kcal/mol. The solvation effect generally weakens the interaction, especially for the charged system. A search of the carboxyl–peptide plane stacking in the PDB databank indicates that parallel stacking but not T-shaped stacking is quite common, and the most probable distance between the two stacking fragments is close to the value predicted by the QM calculations. This work highlights the potential role of carboxyl amide π–π stacking in the stabilization of aspartic acid and glutamic acid in proteins.

An attempt is made to evaluate the dielectric constant of the Trichoderma reesei Cel7B active site. Through kinetic measurements, the pKa value of the catalytic acid E201 is determined. Mutations (away from E201) with net charge changes are introduced to perturb the E201 pKa. It is shown that the mutation with a +1 charge change (including G225R, G230R, and A335R) decreases the pKa of E201, whereas the mutation with a −1 charge change (including Q149E, A222D, G225D, and G230D) increases the pKa. This effect is consistent with the electrostatic interaction between the changed charge and the E201 side chain. The fitting of the experimental data yields an apparent dielectric constant of 25–80. Molecular dynamics simulations with explicit water molecules indicate that the high solvent accessibility of the active site contributes largely to the high dielectric constant. ONIOM calculations show that high dielectric constant benefits the catalysis through decreasing the energy of the transition state relative to that of the enzyme substrate complex.

Parallel and T-shaped stacking interactions of the peptide plane with polar and ionizable amino acid side chains (including aspartic/glutamic acid, asparagine/glutamine, and arginine) are investigated using the quantum mechanical MP2 and CCSD computational methods. It is found that the electrostatic interaction plays an essential role in determining the optimal stacking configurations for all investigated stacking models. For certain complexes, the dispersion interaction also contributes considerably to stacking. In the gas phase, the stacking interaction of the charged system is stronger than that of the neutral system, and T-shaped stacking is generally more preferred than parallel stacking, with the stacking energy in the range of −4 to −18 kcal/mol. The solvation effect overall weakens stacking, especially for the charged system and the T-shaped stacking configurations. In water, the interaction energies of different stacking models are comparable.

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.

A helical nanostructure can be obtained by self-assembly of a diarylethene derivative that bears two malononitrile substitutes in a tetrahydrofuran/water medium. It is revealed that the helical nanostructure changed from helical nanofiber to helical nanotube when the diarylethene monomer changed from the open-ring isomer to the closed-ring isomer upon irradiation with 365 nm ultraviolet light, meanwhile, the helix angle of the nanostructure changed from 50° ± 5° to 75° ± 5°. There is a great possibility that the helical nanofibers and helical nanotubes are assembled from dimers as base units based on theoretical calculation and experimental results.

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.

An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed p-nitrophenyl lactoside hydrolysis. In both the glycosylation and deglycosylation steps, the reaction proceeds in a concerted way, meaning the nucleophilic attack and the glycosidic bond cleavage occur simultaneously. The glycosylation step is rate limiting with a barrier of 18.9 kcal/mol, comparable to the experimental value derived from the kcat measured in this work. The function of four residues R108, Y146, Y170, and D172, which form a hydrogen-bond network involving the substrate, is studied by conservative mutations. The mutants, including R108K, Y146F, Y170F, and D172N, decrease the enzyme activity by about 150–8000-fold. Molecular dynamics simulations show that the mutations disrupt the hydrogen-bond network, cause the substrate to deviate from active binding and hinder either the proton transfer from E201 to O4(+1) or the nucleophilic attack from E196 to C1(−1).

A critical role of the Family 7 cellobiohydrolase (Cel7A) carbohydrate binding domain (CBD) is to bind to a cellulose surface and increase the enzyme concentration on the surface. Several residues of Trichoderma reesei Cel7A CBD, including Y5, N29, Y31, Y32 and Q34, contribute to cellulose binding, as revealed by early experimental studies. To investigate the interactions between these important residues and cellulose, we applied a thermodynamic integration method to calculate the cellulose–Cel7A CBD binding free energy changes caused by Y5A, N29A, Y31A, Y32A and Q34A mutations. The experimental binding trend was successfully predicted, proving the effectiveness of the complex model. For the two polar residue mutants N29A and Q34A, the changes in the electrostatics are comparable to those of van der Waals, while for three Y to A mutants, the free energy differences mainly come from van der Waals interactions. However, in both cases, the electrostatics dominates the interactions between individual residues and cellulose. The side chains of these residues are rigidified after the complex is formed. The binding free energy changes for the two mutants Y5W and Y31W were also determined, and for these the van der Waals interaction was strengthened but the electrostatics was weakened.

A total of 1.1 μs of molecular dynamics (MD) simulations were performed to study the structure and dynamics of protein GB3. The simulation motional amplitude of the loop regions is generally overestimated in comparison with the experimental backbone N−H order parameters S2. Two-state behavior is observed for several residues in these regions, with the minor state population in the range of 3−13%. Further inspection suggests that the (ϕ, ψ) dihedral angles of the minor states deviate from the GB3 experimental values, implying the existence of nonnative states. After fitting the MD trajectories of these residues to the NMR RDCs, the minor state populations are significantly reduced by at least 80%, suggesting that MD simulations are strongly biased toward the minor states, thus overestimating the dynamics of the loop regions. The optimized trajectories produce intra, sequential HN−Hα RDCs and intra 3JHNHα that are not included in the trajectories fitting for these residues that are closer to the experimental data. Unlike GB3, 0.55 μs MD simulations of protein ubiquitin do not show distinctive minor states, and the derived NMR order parameters are better converged. Our findings indicate that the artifacts of the simulations depend on the specific system studied and that one should be cautious interpreting the enhanced dihedral dynamics from long MD simulations.

QM/MD simulations are performed to study mutational effects on the glycosylation step of the oligosaccharide hydrolysis catalyzed by Trichoderma reesei cellobiohydrolase I. The potential of mean force along the reaction pathway is determined by the umbrella sampling method. A detailed mechanism is developed to illustrate the decrease in activity of the mutants. Our calculations demonstrate that (1) the E212Q mutation increases the overall activation barrier by ∼4.0 kcal/mol, while the D214N mutation causes ∼0.4 kcal/mol increase of the barrier, and (2) there is only one transition state identified in the wild type (WT) and D214N mutant, while two transition states exist in the E212Q mutant for the glycosylation process. The results explain the experimental observation that the E212Q mutant loses most of its hydrolysis capability, while the D214N mutant only reduces it slightly compared to the WT. Further analysis suggests that the proton transfer from Glu217 to O4 and the glycosidic bond cleavage between subsites +1 and −1 are concerted, facilitating the subsequent nucleophilic attack of Glu212 on C1′ in subsite −1. Our QM/MD study illustrates the importance of the prearrangement of the active site and provides atomic details of the enzymatic catalytic mechanism.

•A positive correlation is observed between the association constant Kr of the PASC and FP binding.•The slowdown of Cel7B-CD catalysis is directly correlated to its initial activity.•The Cel7B-CD catalytic activity against the insoluble substrate cellulose is determined by the enzyme’s capability in hydrolyzing the soluble substrate.One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24 h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15–35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme’s capability in hydrolyzing the soluble substrate.

A helical nanostructure can be obtained by self-assembly of a diarylethene derivative that bears two malononitrile substitutes in a tetrahydrofuran/water medium. It is revealed that the helical nanostructure changed from helical nanofiber to helical nanotube when the diarylethene monomer changed from the open-ring isomer to the closed-ring isomer upon irradiation with 365 nm ultraviolet light, meanwhile, the helix angle of the nanostructure changed from 50° ± 5° to 75° ± 5°. There is a great possibility that the helical nanofibers and helical nanotubes are assembled from dimers as base units based on theoretical calculation and experimental results.