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

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

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

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

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

Thermal denaturation of the WT FbFP, the single point mutant N124Y, and the triple mutant N107Y-N124Y-M111F.

<p>The fluorescence intensity of the bound FMN is used to monitor the protein denaturation. As can be seen, the mutants have higher percentages of fluorescence at elevated temperature than WT suggesting mutations increase FbFP thermostability.</p

Residues in close contact with I120 (A, B), F107 (C, D), F124 (E, F) and F111 (G, H) are labeled where (A, C, E, G) are from subunit 1 and (B, D, F, H) are from subunit 2.

<p>Residues in close contact with I120 (A, B), F107 (C, D), F124 (E, F) and F111 (G, H) are labeled where (A, C, E, G) are from subunit 1 and (B, D, F, H) are from subunit 2.</p

Melting temperatures of WT and mutant FbFP.

a<p><i>T</i>_m was not determined due to the weak fluorescence of the sample at room temperature.</p

Alkyne Activation by a Porous Silver Coordination Polymer for Heterogeneous Catalysis of Carbon Dioxide Cycloaddition

The widely studied porous coordination polymers, possessing large pores to adsorb waste carbon dioxide gas and further transform it into valuable chemical products, have been attracting research interest, both industrially and academically. The active silver­(I) ions endow the specific alkynophilicity to activate CC bonds of alkyne-containing molecules via π activation. Incorporating catalytic Ag metal sites into the porous frameworks represents a promising approach to construct heterogeneous catalysts that cyclize propargylic alcohols with CO₂, which is highly desirable for the environmentally benign conversion of carbon dioxide to fine chemicals. We report the preparation of porous coordination polymers (PCPs) with active silver sites and efficient silver–silver bond formation by carefully modifying the coordination geometries of the silver sites. The decentralized silver­(I) chains in the porous frameworks enable the efficient conversion of CO₂ and derivatives of acetylene to α-alkylidene cyclic carbonates in a heterogeneous manner. X-ray structure analysis reveals two kinds of substrate molecules positioned within the pores of the framework, which correspond to trapping and activated modes through the multiple interactions with the functional Ag chains. The example of tandem conversion of simple alkynes and carbon dioxide to α-alkylidene cyclic carbonates is also presented. The well-positioned catalytic silver­(I) sites and the crystalline properties of the frameworks facilitated the structural analyses of the intermediates of each catalytic step, providing knowledge of the synergistic nature of the σ and π activation of CC bonds. The successful catalysis of azide–alkyne cycloaddition and synthesis of propargylic alcohols via terminal alkynes could also give another indicator for the activation properties of Ag sites