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Changes in free energy provide valuable information for molecular recognition, including both ligand-receptor binding thermodynamics and kinetics. Umbrella sampling (US), a widely used free energy calculation method, has long been used to explore the dissociation process of ligand-receptor systems and compute binding free energy. In existing publications, the binding free energy computed from the potential of mean force (PMF) with US simulation mostly yielded "ball park" values with experimental data. However, the computed PMF values are highly influenced by factors such as initial conformations and/or trajectories provided, the reaction coordinate, and sampling of conformational space in each US window. These critical factors have rarely been carefully studied. Here we used US to study the guest aspirin and 1-butanol dissociation processes of β-cyclodextrin (β-CD) and an inhibitor SB2 dissociation from a p38α mitogen-activated protein kinase (MAPK) complex. For β-CD, we used three different β-CD conformations to generate the dissociation path with US windows. For p38α, we generated the dissociation pathway by using accelerated molecular dynamics followed by conformational relaxing with short conventional MD, steered MD, and manual pulling. We found that, even for small β-CD complexes, different β-CD conformations altered the height of the PMF, but the pattern of PMF was not affected if the MD sampling in each US window was well-converged. Because changing the macrocyclic ring conformation needs to rotate dihedral angles in the ring, a bound ligand largely restrains the motion of cyclodextrin. Therefore, once a guest is in the binding site, cyclodextrin cannot freely change its initial conformation, resulting in different absolute heights of the PMF, which cannot be overcome by running excessively long MD simulations for each US window. Moreover, if the US simulations were not converged, the important barrier and minimum were missed. For ligand-protein systems, our studies also suggest that the dissociation trajectories modeled by an enhanced sampling method must maintain a natural molecular movement to avoid biased PMF plots when using US simulations.

Summary: The effects of ultrasound (US) pretreatment combined with drying process on storage quality maintenance and bioactive compound accumulation of broccoli stems were investigated. Results indicated that US pretreatment (λ = 8.5 mm) delayed the decline of L* value and ascorbic acid content but inhibited the increase of a* and b* values, browning index, and microbial counts of fresh‐cut broccoli stems. Furthermore, the US application enhanced total phenolics content by activating phenylalanine ammonia‐lyase, cinnamate‐4‐hydroxylase, and 4‐coumarate‐CoA ligase in phenylpropane metabolism. Meanwhile, it also promoted γ‐aminobutyric acid (GABA) accumulation by upregulating activities of diamine oxidase, polyamine oxidase, and aminoaldehyde dehydrogenase in GABA shunt and polyamine degradation pathway, respectively, which in turn, accelerated the biosynthesis of GABA from polyamines and glutamate. In addition, compared with other three drying methods, the combined vacuum freeze drying (VFD) could further retain the sensory attributes and bioactive compounds to a maximum extent in dehydrated broccoli stems. Hence, current findings illustrated that US pretreatment combined with VFD process is beneficial for commercial quality maintenance and nutritional value enhancement, thus providing new perspectives on the comprehensive utilisation of broccoli stems. [ABSTRACT FROM AUTHOR]

Understanding the governing factors of fast or slow inhibitor binding/unbinding assists in developing drugs with preferred kinetic properties. For inhibitors with the same binding affinity targeting different binding sites of the same protein, the kinetic behavior can profoundly differ. In this study, we investigated unbinding kinetics and mechanisms of fast (type-I) and slow (type-II/III) binders of p38α mitogen-activated protein kinase, where the crystal structures showed that type-I and type-II/III inhibitors bind to pockets with different conformations of the Asp-Phe-Gly (DFG) motif. The work used methods that combine conventional molecular dynamics (MD), accelerated molecular dynamics (AMD) simulations, and the newly developed pathway search guided by internal motions (PSIM) method to find dissociation pathways. The study focuses on revealing key interactions and molecular rearrangements that hinder ligand dissociation by using umbrella sampling and post-MD processing to examine changes in free energy during ligand unbinding. As anticipated, the initial dissociation steps all require breaking interactions that appeared in crystal structures of the bound complexes. Interestingly, for type-I inhibitors such as SB2, p38α keeps barrier-free conformational fluctuation in the ligand-bound complex and during ligand dissociation. In contrast, with a type-II/III inhibitor such as BIRB796, with the rearrangements of p38α in its bound state, ligand unbinding features energetically unfavorable protein-ligand concerted movement. Our results also show that the type-II/III inhibitors preferred dissociation pathways through the allosteric channel, which is consistent with an existing publication. The study suggests that the level of required protein rearrangement is one major determining factor of drug binding kinetics in p38α systems, providing useful information for development of inhibitors.

This review discusses the use of molecular modeling tools, together with existing experimental findings, to provide a complete atomic-level description of enzyme dynamics and function. We focus on functionally relevant conformational dynamics of enzymes and the protonation states of substrates. The conformational fluctuations of enzymes usually play a crucial role in substrate recognition and catalysis. Protein dynamics can be altered by a tiny change in a molecular system such as different protonation states of various intermediates or by a significant perturbation such as a ligand association. Here we review recent advances in applying atomistic molecular dynamics (MD) simulations to investigate allosteric and network regulation of tryptophan synthase (TRPS) and protonation states of its intermediates and catalysis. In addition, we review studies using quantum mechanics/molecular mechanics (QM/MM) methods to investigate the protonation states of catalytic residues of β-Ketoacyl ACP synthase I (KasA). We also discuss modeling of large-scale protein motions for HIV-1 protease with coarse-grained Brownian dynamics (BD) simulations.

Four new X-ray structures of tryptophan synthase (TS) crystallized with varying numbers of the amphipathic N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) molecule are presented. These structures show one of the F6 ligands threaded into the tunnel from the β-site and reveal a distinct hydrophobic region. Over this expanse, the interactions between F6 and the tunnel are primarily nonpolar, while the F6 phosphoryl group fits into a polar pocket of the β-subunit active site. Further examination of TS structures reveals that one portion of the tunnel (T1) binds clusters of water molecules, whereas waters are not observed in the nonpolar F6 binding region of the tunnel (T2). MD simulation of another TS structure with an unobstructed tunnel also indicates the T2 region of the tunnel excludes water, consistent with a dewetted state that presents a significant barrier to the transfer of water into the closed β-site. We conclude that hydrophobic molecules can freely diffuse between the α- and β-sites via the tunnel, while water does not. We propose that exclusion of water serves to inhibit reaction of water with the α-aminoacrylate intermediate to form ammonium ion and pyruvate, a deleterious side reaction in the αβ-catalytic cycle. Finally, while most TS structures show βPhe280 partially blocking the tunnel between the α- and β-sites, new structures show an open tunnel, suggesting the flexibility of the βPhe280 side chain. Flexible docking studies and MD simulations confirm that the dynamic behavior of βPhe280 allows unhindered transfer of indole through the tunnel, therefore excluding a gating role for this residue.

The importance of protonation states and proton transfer in pyridoxal 5'-phosphate (PLP)-chemistry can hardly be overstated. Although experimental approaches to investigate pKa values can provide general guidance for assigning proton locations, only static pictures of the chemical species are available. To obtain the overall protein dynamics for the interpretation of detailed enzyme catalysis in this study, guided by information from solid-state NMR, we performed molecular dynamics (MD) simulations for the PLP-dependent enzyme tryptophan synthase (TRPS), whose catalytic mechanism features multiple quasi-stable intermediates. The primary objective of this work is to elucidate how the position of a single proton on the reacting substrate affects local and global protein dynamics during the catalytic cycle. In general, proteins create a chemical environment and an ensemble of conformational motions to recognize different substrates with different protonations. The study of these interactions in TRPS shows that functional groups on the reacting substrate, such as the phosphoryl group, pyridine nitrogen, phenolic oxygen and carboxyl group, of each PLP-bound intermediate play a crucial role in constructing an appropriate molecular interface with TRPS. In particular, the protonation states of the ionizable groups on the PLP cofactor may enhance or weaken the attractions between the enzyme and substrate. In addition, remodulation of the charge distribution for the intermediates may help generate a suitable environment for chemical reactions. The results of our study enhance knowledge of protonation states for several PLP intermediates and help to elucidate their effects on protein dynamics in the function of TRPS and other PLP-dependent enzymes.

The main protease (Mpro) of SARS‐CoV‐2 plays an essential role in the virus life cycle and is considered a key target for therapeutic development. This study explores the inhibition mechanism of SARS‐CoV‐2 Mpro by ebselen, an organoselenium drug that shows potent inhibitory activity. By using a combination of multiple computational methods including molecular docking, molecular dynamics simulations, and density functional theory calculations, the complete covalent inhibition process of ebselen is simulated for the first time. Two possible pathways with different bound conformations of ebselen are identified. The hydrolysis of the enzyme‐ebselen adduct is found to be the rate‐determining step. The simulation results show that the behavior of water molecules at the hydrolysis site is crucial to distinguish the two paths energetically. Our simulations, which are in agreement with existing experimental results, provide a theoretical basis for the rational design and mechanism exploration of ebselen‐based inhibitors. [ABSTRACT FROM AUTHOR]

Initial CO2 electroreduction into CO and its subsequent electroreduction pathways were selected to study the effect of specifically adsorbed halide anions X− (X = F, Cl, Br, I) on CO2 electroreduction activity and product selectivity at Cu(111)/H2O interfaces via DFT calculations. The calculated results show that the presence of halide anions can exert a notable effect on the CO2 adsorption characteristics and that chemically adsorbed CO2 molecules can be formed. Furthermore, the halide-anion-modified Cu(111)/H2O interfaces could significantly enhance the initial CO2 electroreduction into CO activity, which is regarded as the rate-determining step during CO2 electroreduction at clean Cu(111)/H2O interfaces. Analysis of the initial CO2 electroreduction and Volmer reaction pathways showed that the halide-anion-modified Cu(111)/H2O interfaces could suppress the HER and thus improve the CO2 electroreduction activity and product selectivity. It is speculated that the enhanced initial CO2 electroreduction activity at the F−-, Cl−-, Br−-, and I−-modified Cu(111)/H2O interfaces may originate from the decreased work functions and anion radical ·CO2− formations. Simultaneously, we concluded that dimer OCCO formations in the presence of halide anions were more favorable than CHO during CO electroreduction according to the order of I− > Br− > Cl− > F− and could result in the production of C2 product, suggesting an improved CO2 electroreduction product selectivity. The present analyses of electronic structure may explain the more favorable OCCO formations in the order of I− > Br− > Cl− > F−. The present understanding of this effect will provide an improved scientific guideline for the control of CO2 electroreduction pathways and design of more efficient electrocatalysts. [ABSTRACT FROM AUTHOR]

The effects of γ-aminobutyric (GABA) on enzymatic browning, storage quality, membrane and reactive oxygen species (ROS) metabolism in fresh-cut stem lettuce were investigated. The results illustrated that GABA treatment delayed browning degree, polyphenol oxidase (PPO) activity and the expression of LsPPO. Meanwhile, higher chlorophyll and ascorbic acid contents were exhibited in GABA-treated stem lettuce, as well as the slower microbial propagation. Further investigation revealed that exogenous GABA application declined malondialdehyde content, electrolyte leakage and the enzyme activities of membrane metabolism, and the expression levels of related genes were also downregulated. In addition, GABA treatment scavenged ROS and strengthened the enzyme activities of ROS metabolism, as well as the expression levels of corresponding genes. Taken together, these findings implied that the repressed enzymatic browning and microbial propagation in GABA-treated stem lettuce were due to the inhibition of ROS accumulation, enhancement of membrane stability and increased resistance to oxidation. • Exogenous GABA inhibited the enzymatic browning of fresh-cut stem lettuce. • GABA treatment maintained the quality in fresh-cut stem lettuce. • GABA application inhibited the microbial propagation in fresh-cut stem lettuce. • GABA treated-fresh-cut stem lettuce exhibited higher membrane stability. • GABA treatment enhanced the antioxidant capacity in fresh-cut stem lettuce. [ABSTRACT FROM AUTHOR]

[Display omitted] • Glutamate treatment alleviated whitening development in fresh-cut carrots. • Glutamate maintained total carotenoid content in fresh-cut carrots. • Glutamate stimulated a rapid accumulation of GABA in fresh-cut carrots. • Glutamate treatment increased phenolics content of fresh-cut carrots. • ROS scavenging system was activated by glutamate treatment in fresh-cut carrots. The effects of exogenous glutamate treatment on the quality attributes, γ-aminobutyric acid (GABA) shunt, phenylpropanoid pathway, and antioxidant capacity of fresh-cut carrots were investigated. Results showed that glutamate treatment suppressed the increases in lightness and whiteness values, inhibited the degradation of total carotenoids and maintained better flavor and taste in fresh-cut carrots. Moreover, glutamate treatment rapidly promoted the activities of glutamate decarboxylase and GABA transaminase, thus improving the GABA content. It also significantly enhanced the activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumarate coenzyme A ligase and promoted the accumulation of total phenolics as well as the main individual phenolic compounds, including chlorogenic and caffeic acid. In addition, glutamate application activated the reactive oxygen system-related enzyme including peroxidase, superoxide dismutase, ascorbate peroxidase, and catalase activities to maintain higher antioxidant capacity in fresh-cut carrots. These results demonstrated that exogenous glutamate treatment maintained better nutritional quality and alleviated color deterioration by accelerating the accumulation of GABA and phenolics and enhancing the antioxidant capacity in fresh-cut carrots. [ABSTRACT FROM AUTHOR]

Initial CO 2 electroreduction into CO and its subsequent electroreduction pathways were selected to study the effect of specifically adsorbed halide anions X - (X = F, Cl, Br, I) on CO 2 electroreduction activity and product selectivity at Cu(111)/H 2 O interfaces via DFT calculations. The calculated results show that the presence of halide anions can exert a notable effect on the CO 2 adsorption characteristics and that chemically adsorbed CO 2 molecules can be formed. Furthermore, the halide-anion-modified Cu(111)/H 2 O interfaces could significantly enhance the initial CO 2 electroreduction into CO activity, which is regarded as the rate-determining step during CO 2 electroreduction at clean Cu(111)/H 2 O interfaces. Analysis of the initial CO 2 electroreduction and Volmer reaction pathways showed that the halide-anion-modified Cu(111)/H 2 O interfaces could suppress the HER and thus improve the CO 2 electroreduction activity and product selectivity. It is speculated that the enhanced initial CO 2 electroreduction activity at the F - -, Cl - -, Br - -, and I - -modified Cu(111)/H 2 O interfaces may originate from the decreased work functions and anion radical · CO 2 - formations. Simultaneously, we concluded that dimer OCCO formations in the presence of halide anions were more favorable than CHO during CO electroreduction according to the order of I - > Br - > Cl - > F - and could result in the production of C 2 product, suggesting an improved CO 2 electroreduction product selectivity. The present analyses of electronic structure may explain the more favorable OCCO formations in the order of I - > Br - > Cl - > F - . The present understanding of this effect will provide an improved scientific guideline for the control of CO 2 electroreduction pathways and design of more efficient electrocatalysts.