Inactivation of polyphenol oxidase by low intensity DC field: Experiment and mechanism analysis via molecular dynamics simulation and molecular docking.

In this study, inactivation of mushroom polyphenol oxidase (PPO) by low intensity direct current (DC) electric field and its molecular mechanism were investigated. In the experiments under 3 V/cm, 5 V/cm, 7 V/cm and 9 V/cm electric fields, PPOs were all completely inactivated after different exposure times. Under 1 V/cm, a residual activity of 11.88 % remained. The inactivation kinetics confirms to Weibull model. Under 1-7 V/cm, n value closes to a constant about 1.3. The structural analysis of PPO under 3 V/cm and 5 V/cm by fluorescence emission spectroscopy and molecular dynamics (MD) simulation showed that the tertiary structure was slightly changed with increased radius of gyration, higher potential energy and rate of C-alpha fluctuation. After exposure to the electric field, most of the hydrophobic tryptophan (TRP) residues turned to the hydrophilic surface, resulting the fluorescence red-shifted and quenched. Molecular docking indicated that the receptor binding domain of catechol in PPO was changed. PPO under electric field was MD simulated the first time, revealing the changing mechanism of the electric field itself on PPO, a binuclear copper enzyme, which has a metallic center. All these suggest that the low intensity DC electric field would be a promising option for enzymatic browning inhibition or even enzyme activity inactivation.

Multiscale approach to the characterization of the interfacial properties of micellar casein and whey protein blends and their effects on recombined dairy creams.

In this study, whipped cream with blends of micellar casein (MCN) and whey protein (WPI) in different ratios were prepared to investigate the role of protein interfacial behavior in determining foam properties at multiple scales, using theoretical modeling, and microscopic and macroscopic analysis. Fluid force microscopy has been used for the first time as a more realistic and direct means of analyzing interfaces properties in multiphase systems. The adsorption kinetics showed that the interfacial permeability constant of WPI (4.24 × 10-4 s-1) was significantly higher than that of the MCN (2.97 × 10-4 s-1), and the WPI interfacial layer had a higher modulus of elasticity (71.38 mN/m) than that of the MCN (47.89 mN/m). This model was validated via the mechanical analysis of the fat globules in real emulsions. The WPI-stabilized fat globule was found to have a higher Young's modulus (219.67 Pa), which contributes to the integrity of its fat globule morphology. As the ratio of MCN was increased in the sample, however, both the interfacial modulus and Young's modulus decreased. Moreover, the rate of partial coalescence was found to increase, a phenomenon that decreased the stability of the emulsion and increased the rate of aeration. The mechanical analysis also revealed a higher level of adhesion between MCN-stabilized fat globule (25.16 nN), which increased fat globule aggregation and emulsion viscosity, while improving thixotropic recovery. The synergistic effect of the blended MCN and WPI provided the highest overrun, at 194.53 %. These studies elucidate the role of the interfacial behavior of proteins in determining the quality of whipped cream and provide ideas for the application of proteins in multiphase systems.

Impact of chickpea hummus on postprandial blood glucose, insulin and gut hormones in healthy humans combined with mechanistic studies of food structure, rheology and digestion kinetics.

Slowing the rate of carbohydrate digestion leads to low postprandial glucose and insulin responses, which are associated with reduced risk of type 2 diabetes. There is increasing evidence that food structure plays a crucial role in influencing the bioaccessibility and digestion kinetics of macronutrients. The aims of this study were to compare the effects of two hummus meals, with different degrees of cell wall integrity, on postprandial metabolic responses in relation to the microstructural and rheological characteristics of the meals. A randomised crossover trial in 15 healthy participants was designed to compare the acute effect of 27 g of starch, provided as hummus made from either intact chickpea cells (ICC) or ruptured chickpea cells (RCC), on postprandial metabolic responses. In vitro starch digestibility, microstructural and rheological experiments were also conducted to evaluate differences between the two chickpea hummus meals. Blood insulin and GIP concentrations were significantly lower (P < 0.02, P < 0.03) after the consumption of the ICC meal than the meal containing RCC. In vitro starch digestion for 90 min was slower in ICC than in RCC. Microscopic examination of hummus samples digested in vitro for 90 min revealed more intact chickpea cells in ICC compared to the RCC sample. Rheological experiments showed that fracture for ICC hummus samples occurred at smaller strains compared to RCC samples. However, the storage modulus for ICC was higher than RCC, which may be explained by the presence of intact cells in ICC. Food structure can affect the rate and extent of starch bioaccessibility and digestion and may explain the difference in the time course of metabolic responses between meals. The rheological properties were measured on the two types of meals before ingestion, showing significant differences that may point to different breakdown mechanisms during subsequent digestion. This trial was registered at clinicaltrial.gov as NCT03424187.

Droplet size dependency and spatial heterogeneity of lipid oxidation in whey protein isolate-stabilized emulsions.

Spatiotemporal assessment of lipid and protein oxidation is key for understanding quality deterioration in emulsified food products containing polyunsaturated fatty acids. In this work, we first mechanistically validated the use of the lipid oxidation-sensitive fluorophore BODIPY 665/676 as a semi-quantitative marker for local peroxyl radical formation. Next, we assessed the impact of microfluidic and colloid mill emulsification (respectively producing mono- and polydisperse droplets) on local protein and lipid oxidation kinetics in whey protein isolate (WPI)-stabilized emulsions. We further used BODIPY 581/591 C11 and CAMPO-AFDye 647 as colocalisation markers for lipid and protein oxidation. The polydisperse emulsions showed an inverse relation between droplet size and lipid oxidation rate. Further, we observed less protein and lipid oxidation occurring in similar sized droplets in monodisperse emulsions. This observation was linked to more heterogeneous protein packing at the droplet surface during colloid mill emulsification, resulting in larger inter-droplet heterogeneity in both protein and lipid oxidation. Our findings indicate the critical roles of emulsification methods and droplet sizes in understanding and managing lipid oxidation.

Pathways of amyloid fibril formation and protein aggregation.

The main cause of many neurodegenerative diseases and systemic amyloidoses is protein and peptide aggregation and the formation of amyloid fibrils. The study of aggregation mechanisms, the discovery and description of aggregate structures, and a comprehensive understanding of the molecular mechanisms of amyloid formation are of great importance for the diagnostic processes at the molecular level and for the development of therapeutic strategies to counter aggregation-associated disorders. Given that understanding protein misfolding phenomena is directly related to the protein folding process, we will briefly explain the protein folding mechanism and then discuss the important factors involved in protein aggregation. In the following, we review different mechanisms of amyloid formation and finally represent the current knowledge on how amyloid fibrils are formed based on kinetic and thermodynamic factors.

Construction and mechanistic insights of a novel ZnO functionalized rGO composite for efficient adsorption and reduction of Cr(VI).

A series of ZnO decorated reduced graphene oxide (rGO) (ZnrGOx) with different doping ratios were synthesized by the alkaline hydrothermal method using graphene oxide (GO) and Zn(NO3)2·6H2O as precursors, and subsequently used for the adsorption study of Cr(VI) in water. The morphology, crystalline phase structure, and surface elemental properties of ZnrGOx composites were revealed by XRD, SEM, BET, FT-IR, and XPS characterizations. The results showed that ZnO nanoparticles can be clearly seen on the surface of layered rGO. Meanwhile, as the doping rate increased, the C = C double bonds were broken and more carboxylic acid groups formed in ZnrGOx. In addition, the ZnrGO0.1 composite had the most excellent adsorption performance and good stability, and reusability. The adsorption removal rate of Cr(VI) can reach 99%, and the maximum adsorption amount of Cr(VI) was 68.9655 mg/g in 3 h. The isothermal and kinetic model simulations showed that Cr(VI) adsorption on ZnrGO0.1 composite is a chemical adsorption process, spontaneous and endothermic. Based on the concentrations of different valence states of Cr in the solid and liquid phases, 40% of Cr(VI) was reduced to Cr(III) on the surface of ZnrGO0.1 composite. Moreover, the adsorption-reduction mechanisms of Cr(VI) on ZnrGO0.1 composite were further elucidated. The ZnrGO0.1 composite manifested great potential as an efficient adsorbent for Cr(VI) removal.

A Pharmacological perspective on the temporal properties of sweeteners.

Several non-caloric sweeteners exhibit a delay in sweetness onset and a sweetness linger after sampling. These temporal properties are thought to be the result of non-specific interactions with cell membranes and proteins in the oral cavity. Data and analysis presented in this report also support the potential involvement of receptor affinity and binding kinetics to this phenomenon. In general, affected sweeteners exhibit distinctly higher binding affinity compared to carbohydrate sweeteners, which do not have temporal issues. In addition, binding kinetic simulations illustrate much slower receptor binding association and dissociation kinetics for a set of non-caloric sweeteners presenting temporal issues, in comparison to carbohydrate sweeteners. So, the higher affinity of some non-caloric sweeteners, dictating lower use levels, and affecting binding kinetics, could contribute to their delay and linger in sweetness perception. Simple pharmacology principles could explain, at least in part, some of the temporal issues of sweeteners.

Application of UV LEDs to inactivate antibiotic resistant bacteria: Kinetics, efficiencies, and reactivations.

Unregulated antibiotic use has led to the proliferation of antibiotic-resistant bacteria (ARB) in aquatic environments. Ultraviolet light-emitting diodes (UV LEDs) have evolved as an innovative technology for inactivating microorganisms offering several advantages over traditional mercury lamps. This research concentrated on utilizing UV LEDs with three distinct wavelengths (265 nm, 275 nm, and 285 nm) to inactivate E. coli DH10ß encoding the ampicillin-resistant blaTEM-1 gene in its plasmid. Non-linear models, such as Geeraerd's and Weibull, provided more accurate characterization of the inactivation profiles than the traditional log-linear model due to the incorporation of both biological mechanisms and a deterministic approach within non-linear models. The inactivation rates of ARB were higher than antibiotic-sensitive bacteria (ASB) when subjected to UV LEDs. The highest inactivation rates were observed when all microorganisms were exposed to 265 nm. Photoreactivation emerged as the primary mechanism responsible for repairing DNA damage induced by UV LEDs. 285 nm showed the highest reactivation efficiencies for ARB under different fluences. At higher fluences, both 265 and 275 nm displayed similar effectiveness in suppressing reactivation, while at lower fluences, 275 nm exhibited better efficacies in controlling the reactivation. Therefore, the inhibition of reactivation was influenced by the extent of damage incurred to both DNA and enzymes. In nutrient-poor media (0.9 % NaCl), ASB did not exhibit any reactivation potential. However, the addition of Luria-Bertani (LB) broth promoted the reactivation of ASB. Lower fluence rate was more beneficial at 265 nm whereas higher fluence rates were more effective for longer wavelengths. The inactivation of ARB was enhanced by dissolved organic carbon (DOC) at low fluences. However, the removal of ARB was reduced due to the presence of DOC at higher fluences. The highest energy demand for ARB inactivation was reported at 285 nm. ENVIRONMENTAL IMPLICATION: The excessive and unregulated utilization of antibiotics has emerged as a significant issue for public health. This paper presents a comprehensive analysis of the effectiveness of UV LEDs, an emerging technology, in the inactivation of antibiotic-resistant bacteria (ARB). This research paper explores the kinetics of UV LEDs with different wavelengths to inactivate ARB along with the reactivation efficiencies. This research work also explores the impact and relevant mechanisms of the impact of dissolved organic carbon (DOC) on the inactivation of ARB by UV LEDs.

Efficient removal of crystal violet dye from water using zinc ferrite-polyaniline nanocomposites.

Nanomaterials are widely employed in wastewater treatment, among which nanoferrites and their composites hold significant prominence. This study adopts a green approach to synthesize zinc ferrite nanoparticles, subsequently integrating them with polyaniline (PANI) to fabricate the ZnFe2O4-PANI nanocomposite. Characterization of the prepared ZnFe2O4-PANI nanocomposite was conducted using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopic (SEM) techniques. Using Scherrer's equation, the crystallite size of the synthesized zinc ferrite nanoparticles was found to be 17.67 nm. SEM micrographs of the ZnFe2O4-PANI nanocomposite revealed that in situ polymerization of ZnFe2O4 with polyaniline transforms the amorphous surface morphology of the polymer into a homogeneous nanoparticle structure. The adsorption of crystal violet (CV) dye onto the surface of the ZnFe2O4-PANI nanocomposite depends on pH, adsorbent dosage, temperature, concentration levels and duration. The Langmuir adsorption model fitted the data well, indicating adherence to a pseudo-second-order kinetic pattern. Thermodynamic values ΔG°, ΔH° and ΔS° indicated that the adsorption process occurred spontaneously. Advantages and disadvantages of the technique have also been highlighted. Mechanism of adsorption is discussed. From the obtained results, it is evident that the ZnFe2O4-PANI nanocomposite holds promise as a sorbent for the removal of dye from wastewater.

Removal of lead ions from aqueous solution by modified nanocellulose.

Heavy metals significantly impact the environment due to their non-biodegradable, toxic, and carcinogenic behaviors. Lead contaminants impose severe health impacts on humans and the water environment. Therefore, eco-friendly and efficient lead ion removal practices such as nanotechnology are an urgent requirement for the abatement of lead pollution. In the present study, nanocellulose was synthesized from the cotton straw residue using chemical methods and modified with titanium dioxide to form a nanocomposite. The nanocomposite synthesized was characterized by using FTIR, XRD, FESEM, and BET. FTIR results noticed peaks at 1648.43 and 1443.57 cm-1 for cellulose and Ti-O-Ti bonding at 505.02 cm-1. The nanocomposite was noticed to be disordered and irregular in shape. The nanocomposite has particle sizes of 83 nm. The nanocomposite crystalline particle had 65% anatase and 32% rutile phases observed from the XRD result. BET results show that the surface area of nanocellulose increases after surface modification from 25.692 to 42.510 m2/g. The adsorption capacity of the nanocomposite was 0.552 mg/g was noticed. The Elovich kinetic and Baudu isotherms are the best-fitted models for lead ion adsorption. Thermodynamic parameters resulted in Gibbs free energy decreasing with temperature. This study revealed that modified cellulosic adsorbents efficiently absorbed lead ions derived from cotton straws.

A Guide to Computational Cotranscriptional Folding Featuring the SRP RNA.

Although RNA molecules are synthesized via transcription, little is known about the general impact of cotranscriptional folding in vivo. We present different computational approaches for the simulation of changing structure ensembles during transcription, including interpretations with respect to experimental data from literature. Specifically, we analyze different mutations of the E. coli SRP RNA, which has been studied comparatively well in previous literature, yet the details of which specific metastable structures form as well as when they form are still under debate. Here, we combine thermodynamic and kinetic, deterministic, and stochastic models with automated and visual inspection of those systems to derive the most likely scenario of which substructures form at which point during transcription. The simulations do not only provide explanations for present experimental observations but also suggest previously unnoticed conformations that may be verified through future experimental studies.

Deciphering heterogeneous enzymatic surface reactions on xylan using surface plasmon resonance spectroscopy.

Xylans' unique properties make it attractive for a variety of industries, including paper, food, and biochemical production. While for some applications the preservation of its natural structure is crucial, for others the degradation into monosaccharides is essential. For the complete breakdown, the use of several enzymes is required, due to its structural complexity. In fact, the specificity of enzymatically-catalyzed reactions is guided by the surface, limiting or regulating accessibility and serving structurally encoded input guiding the actions of the enzymes. Here, we investigate enzymes at surfaces rich in xylan using surface plasmon resonance spectroscopy. The influence of diffusion and changes in substrate morphology is studied via enzyme surface kinetics simulations, yielding reaction rates and constants. We propose kinetic models, which can be applied to the degradation of multilayer biopolymer films. The most advanced model was verified by its successful application to the degradation of a thin film of polyhydroxybutyrate treated with a polyhydroxybutyrate-depolymerase. The herein derived models can be employed to quantify the degradation kinetics of various enzymes on biopolymers in heterogeneous environments, often prevalent in industrial processes. The identification of key factors influencing reaction rates such as inhibition will contribute to the quantification of intricate dynamics in complex systems.

Removal of Azo Dyes from Water Using Natural Luffa cylindrica as a Non-Conventional Adsorbent.

Reducing high concentrations of pollutants such as heavy metals, pesticides, drugs, and dyes from water is an emerging necessity. We evaluated the use of Luffa cylindrica (Lc) as a natural non-conventional adsorbent to remove azo dye mixture (ADM) from water. The capacity of Lc at three different doses (2.5, 5.0, and 10.0 g/L) was evaluated using three concentrations of azo dyes (0.125, 0.250, and 0.500 g/L). The removal percent (R%), maximum adsorption capacity (Qm), isotherm and kinetics adsorption models, and pH influence were evaluated, and Fourier-transform infrared spectroscopy and scanning electron microscopy were performed. The maximum R% was 70.8% for 10.0 g L-1Lc and 0.125 g L-1 ADM. The Qm of Lc was 161.29 mg g-1. Adsorption by Lc obeys a Langmuir isotherm and occurs through the pseudo-second-order kinetic model. Statistical analysis showed that the adsorbent dose, the azo dye concentration, and contact time significantly influenced R% and the adsorption capacity. These findings indicate that Lc could be used as a natural non-conventional adsorbent to reduce ADM in water, and it has a potential application in the pretreatment of wastewaters.

Exploring the Potential of Biochar Derived from Chinese Herbal Medicine Residue for Efficient Removal of Norfloxacin.

One-step carbonization was explored to prepare biochar using the residue of a traditional Chinese herbal medicine, Atropa belladonna L. (ABL), as the raw material. The resulting biochar, known as ABLB4, was evaluated for its potential as a sustainable material for norfloxacin (NOR) adsorption in water. Subsequently, a comprehensive analysis of adsorption isotherms, kinetics, and thermodynamics was conducted through batch adsorption experiments. The maximum calculated NOR adsorption capacity was 252.0 mg/g at 298 K, and the spontaneous and exothermic adsorption of NOR on ABLB4 could be better suited to a pseudo-first-order kinetic model and Langmuir model. The adsorption process observed is influenced by pore diffusion, π-π interaction, electrostatic interaction, and hydrogen bonding between ABLB4 and NOR molecules. Moreover, the utilization of response surface modeling (RSM) facilitated the optimization of the removal efficiency of NOR, yielding a maximum removal rate of 97.4% at a temperature of 304.8 K, an initial concentration of 67.1 mg/L, and a pH of 7.4. Furthermore, the biochar demonstrated favorable economic advantages, with a payback of 852.5 USD/t. More importantly, even after undergoing five cycles, ABLB4 exhibited a consistently high NOR removal rate, indicating its significant potential for application in NOR adsorption.

Zinc N,N-bis(2-picolyl)amine Chelates Show Substitution-Dependent Cleavage of Phosphodiesters in Models as Well as of PNAzyme-RNA Bulges.

PNAzymes are a group of artificial enzymes which show promising results in selective and efficient cleavage of RNA targets. In the present study, we introduce a series of metal chelating groups based on N,N-bis(2-picolyl) groups (parent, 6-methyl and 6-amino substituted) as the active sites of novel PNAzymes. An improved synthetic route for the 6-amino analogues is described. The catalytic activity of the chelating groups for cleaving phosphodiesters were assessed with the model substrate 2-hydroxypropyl p-nitrophenyl phosphate (HPNPP), confirming that the zinc complexes have the reactivity order of parent < 2-methyl < 2-amino. The three ligands were conjugated to a PNA oligomer to form three PNAzymes which showed the same order of reactivity and some sensitivity to the size of the RNA bulge designed into the catalyst-substrate complex. This work demonstrates that the kinetic activity observed for the model substrate HPNPP could be translated onto the PNAzymes, but that more reactive Zn complexes are required for such PNAzymes to be viable therapeutic agents.

Utilizing olive leaves biomass as an efficient adsorbent for ciprofloxacin removal: characterization, isotherm, kinetic, and thermodynamic analysis.

Olive leaves were utilized to produce activated biomass for the removal of ciprofloxacin (CIP) from water. The raw biomass (ROLB) was activated with sodium hydroxide, phosphoric acid, and Dead Sea water to create co-precipitated adsorbent (COLB) with improved adsorption performance. The characteristics of the ROLB and COLB were examined using SEM images, BET surface area analyzer, and ATR-FTIR spectroscopy. COLB has a BET surface area of 7.763 m2/g, markedly higher than ROLB's 2.8 m2/g, indicating a substantial increase in adsorption sites. Through investigations on operational parameters, the optimal adsorption efficiency was achieved by COLB is 77.9% within 60 min, obtained at pH 6, and CIP concentration of 2 mg/mL. Isotherm studies indicated that both Langmuir and Freundlich models fit the adsorption data well for CIP onto ROLB and COLB, with R2 values exceeding 0.95, suggesting effective monolayer and heterogeneous surface adsorption. The Langmuir model revealed maximum adsorption capacities of 636 mg/g for ROLB and 1243 mg/g for COLB, highlighting COLB's superior adsorption capability attributed to its enhanced surface characteristics post-modification. Kinetic data fitting the pseudo-second-order model with R2 of 0.99 for ROLB and 1 for COLB, along with a higher calculated qe for COLB, suggest its modified surface provides more effective binding sites for CIP, enhancing adsorption capacity. Thermodynamic analysis revealed that the adsorption process is spontaneous (∆Go < 0), and exothermic (∆Ho < 0), and exhibits a decrease in randomness (∆So < 0) as the process progresses. The ΔH° value of 10.6 kJ/mol for ROLB signifies physisorption, whereas 35.97 kJ/mol for COLB implies that CIP adsorption on COLB occurs through a mixed physicochemical process.

Unveiling the potential of machine learning in cost-effective degradation of pharmaceutically active compounds: A stirred photo-reactor study.

In this study, neural networks and support vector regression (SVR) were employed to predict the degradation over three pharmaceutically active compounds (PhACs): Ibuprofen (IBP), diclofenac (DCF), and caffeine (CAF) within a stirred reactor featuring a flotation cell with two non-concentric ultraviolet lamps. A total of 438 datapoints were collected from published works and distributed into 70% training and 30% test datasets while cross-validation was utilized to assess the training reliability. The models incorporated 15 input variables concerning reaction kinetics, molecular properties, hydrodynamic information, presence of radiation, and catalytic properties. It was observed that the Support Vector Regression (SVR) presented a poor performance as the ε hyperparameter ignored large error over low concentration levels. Meanwhile, the Artificial Neural Networks (ANN) model was able to provide rough estimations on the expected degradation of the pollutants without requiring information regarding reaction rate constants. The multi-objective optimization analysis suggested a leading role due to ozone kinetic for a rapid degradation of the contaminants and most of the results required intensification with hydrogen peroxide and Fenton process. Although both models were affected by accuracy limitations, this work provided a lightweight model to evaluate different Advanced Oxidation Processes (AOPs) by providing general information regarding the process operational conditions as well as know molecular and catalytic properties.

Tracking the Cellular Degradation of Silver Nanoparticles: Development of a Generic Kinetic Model.

Understanding the degradation of nanoparticles (NPs) after crossing the cell plasma membrane is crucial in drug delivery designs and cytotoxicity assessment. However, the key factors controlling the degradable kinetics remain unclear due to the absence of a quantification model. In this study, subcellular imaging of silver nanoparticles (AgNPs) was used to determine the intracellular transfer of AgNPs, and single particle ICP-MS was utilized to track the degradation process. A cellular kinetic model was subsequently developed to describe the uptake, transfer, and degradation behaviors of AgNPs. Our model demonstrated that the intracellular degradation efficiency of AgNPs was much higher than that determined by mimicking testing, and the degradation of NPs was highly influenced by cellular factors. Specifically, deficiencies in Ca or Zn primarily decreased the kinetic dissolution of NPs, while a Ca deficiency also resulted in the retardation of NP transfer. The biological significance of these kinetic parameters was strongly revealed. Our model indicated that the majority of internalized AgNPs dissolved, with the resulting ions being rapidly depurated. The release of Ag ions was largely dependent on the microvesicle-mediated route. By changing the coating and size of AgNPs, the model results suggested that size influenced the transfer of NPs into the degradation process, whereas coating affected the degradation kinetics. Overall, our developed model provides a valuable tool for understanding and predicting the impacts of the physicochemical properties of NPs and the ambient environment on nanotoxicity and therapeutic efficacy.

Single-Molecule FRET Analyses of NMDA Receptors.

Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies.

Understanding the impact of binding free energy and kinetics calculations in modern drug discovery.

INTRODUCTION: For rational drug design, it is crucial to understand the receptor-drug binding processes and mechanisms. A new era for the use of computer simulations in predicting drug-receptor interactions at an atomic level has begun with remarkable advances in supercomputing and methodological breakthroughs. AREAS COVERED: End-point free energy calculation methods such as Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) or Molecular-Mechanics/Generalized Born Surface Area (MM/GBSA), free energy perturbation (FEP), and thermodynamic integration (TI) are commonly used for binding free energy calculations in drug discovery. In addition, kinetic dissociation and association rate constants (koff and kon) play critical roles in the function of drugs. Nowadays, Molecular Dynamics (MD) and enhanced sampling simulations are increasingly being used in drug discovery. Here, the authors provide a review of the computational techniques used in drug binding free energy and kinetics calculations. EXPERT OPINION: The applications of computational methods in drug discovery and design are expanding, thanks to improved predictions of the binding free energy and kinetic rates of drug molecules. Recent microsecond-timescale enhanced sampling simulations have made it possible to accurately capture repetitive ligand binding and dissociation, facilitating more efficient and accurate calculations of ligand binding free energy and kinetics.