Computational Study of Peptide Plane Stacking with Polar and Ionizable Amino Acid Side Chains

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

Liquid Crystalline Phase of G-Tetrad DNA for NMR Study of Detergent-Solubilized Proteins

Liquid Crystalline Phase of G-Tetrad DNA for NMR Study of Detergent-Solubilized Protein

A Mechanistic Study of Trichoderma reesei Cel7B Catalyzed Glycosidic Bond Cleavage

An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed <i>p</i>-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 <i>k</i>_cat 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)

The Slowdown of the Endoglucanase <i>Trichoderma reesei</i> Cel5A-Catalyzed Cellulose Hydrolysis Is Related to Its Initial Activity

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 <i>Tr</i>. 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 <i>K</i>_r 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 <i>K</i>_r 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 <i>p</i>-nitrophenyl cellobioside

The Impact of Hydrogen Bonding on Amide ¹H Chemical Shift Anisotropy Studied by Cross-Correlated Relaxation and Liquid Crystal NMR Spectroscopy

Site-specific ¹H chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide moieties in the B3 domain of protein G (GB3). Experimental input data include residual chemical shift anisotropy (RCSA), measured in six mutants that align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension, and cross-correlated relaxation rates between the ¹H^N CSA tensor and either the ¹H−¹⁵N, the ¹H−¹³C′, or the ¹H−¹³C^α dipolar interactions. Analyses with the assumption that the ¹H^N CSA tensor is symmetric with respect to the peptide plane (three-parameter fit) or without this premise (five-parameter fit) yield very similar results, confirming the robustness of the experimental input data, and that, to a good approximation, one of the principal components orients orthogonal to the peptide plane. ¹H^N CSA tensors are found to deviate strongly from axial symmetry, with the most shielded tensor component roughly parallel to the N−H vector, and the least shielded component orthogonal to the peptide plane. DFT calculations on pairs of <i>N</i>-methyl acetamide and acetamide in H-bonded geometries taken from the GB3 X-ray structure correlate with experimental data and indicate that H-bonding effects dominate variations in the ¹H^N CSA. Using experimentally derived ¹H^N CSA tensors, the optimal relaxation interference effect needed for narrowest ¹H^N TROSY line widths is found at ∼1200 MHz

Quinary Interactions Weaken the Electric Field Generated by Protein Side-Chain Charges in the Cell-like Environment

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 <i>E. coli</i> 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

Direct Observation of CH/CH van der Waals Interactions in Proteins by NMR

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 ^vdw<i>J</i>_CC-coupling between protein aliphatic side chain groups. Specifically, ^vdw<i>J</i>_CC-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 <i>J</i>-coupling is correlated with the exchange-repulsion term of van der Waals interaction. NMR detection of ^vdw<i>J</i>_CC-coupling offers a new tool to characterize such interactions in proteins

Carboxyl–Peptide Plane Stacking Is Important for Stabilization of Buried E305 of Trichoderma reesei Cel5A

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 <i>T</i>₅₀ 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

Protein Apparent Dielectric Constant and Its Temperature Dependence from Remote Chemical Shift Effects

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, ¹H^N 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 ε_a of 8.6 ± 0.8 at 298 K. These CSPs, and their derived ε_a value, scale strongly with temperature. For example, CSPs at 313 K are about ∼30% smaller than those at 278 K, corresponding to an effective ε_a 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 ε_a

Engineering a More Thermostable Blue Light Photo Receptor <i>Bacillus subtilis</i> YtvA LOV Domain by a Computer Aided Rational Design Method

<div><p>The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the <i>T</i>_m of the flavin mononucleotide based fluorescent protein <i>Bacillus Subtilis</i> YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.</p></div