Study on the interaction mechanism of whey protein isolate with phosphatidylcholine: By multispectral methods and molecular docking.

The elucidation of the interaction mechanism between phospholipids and milk proteins within emulsions is pivotal for comprehending the properties of infant formula fat globules. In this study, multispectral methods and molecular docking were employed to explore the relationship between phosphatidylcholine (PC) and whey protein isolate (WPI). Observations indicate that the binding constant, alongside thermodynamic parameters, diminishes as temperature ascends, hinting at a predominantly static quenching mechanism. Predominantly, van der Waals forces and hydrogen bonds constitute the core interactions between WPI and PC. This assertion is further substantiated by Fourier transform infrared spectroscopy, which verifies PC's influence on WPI's secondary structure. A detailed assessment of thermodynamic parameters coupled with molecular docking reveals that PC predominantly adheres to specific sites within α-lactalbumin, ß-lactoglobulin, and bovine serum albumin, propelled by a synergy of hydrophobic interactions, hydrogen bonding, and van der Waals forces, with binding energies noted at -5.59, -6.71, and -7.85 kcal/mol, respectively. An increment in PC concentration is observed to amplify the emulsification properties of WPI whilst concurrently diminishing the zeta potential. This study establishes a theoretical foundation for applying the PC-WPI interaction mechanism in food.

System-agnostic prediction of pharmaceutical excipient miscibility via computing-as-a-service and experimental validation.

We applied computing-as-a-service to the unattended system-agnostic miscibility prediction of the pharmaceutical surfactants, Vitamin E TPGS and Tween 80, with Copovidone VA64 polymer at temperature relevant for the pharmaceutical hot melt extrusion process. The computations were performed in lieu of running exhaustive hot melt extrusion experiments to identify surfactant-polymer miscibility limits. The computing scheme involved a massively parallelized architecture for molecular dynamics and free energy perturbation from which binodal, spinodal, and mechanical mixture critical points were detected on molar Gibbs free energy profiles at 180 °C. We established tight agreement between the computed stability (miscibility) limits of 9.0 and 10.0 wt% vs. the experimental 7 and 9 wt% for the Vitamin E TPGS and Tween 80 systems, respectively, and identified different destabilizing mechanisms applicable to each system. This paradigm supports that computational stability prediction may serve as a physically meaningful, resource-efficient, and operationally sensible digital twin to experimental screening tests of pharmaceutical systems. This approach is also relevant to amorphous solid dispersion drug delivery systems, as it can identify critical stability points of active pharmaceutical ingredient/excipient mixtures.

The effect of hydrostatic pressure on lipid membrane lateral structure.

High pressure is both an environmental challenge to which deep sea biology has to adapt, and a highly sensitive thermodynamic tool that can be used to trigger structural changes in biological molecules and assemblies. Lipid membranes are amongst the most pressure sensitive biological assemblies and pressure can have a large influence on their structure and properties. In this chapter, we will explore the use of high pressure small angle X-ray diffraction and high pressure microscopy to measure and quantify changes in the lateral structure of lipid membranes under both equilibrium high pressure conditions and in response to pressure jumps.

Force Field X: A computational microscope to study genetic variation and organic crystals using theory and experiment.

Force Field X (FFX) is an open-source software package for atomic resolution modeling of genetic variants and organic crystals that leverages advanced potential energy functions and experimental data. FFX currently consists of nine modular packages with novel algorithms that include global optimization via a many-body expansion, acid-base chemistry using polarizable constant-pH molecular dynamics, estimation of free energy differences, generalized Kirkwood implicit solvent models, and many more. Applications of FFX focus on the use and development of a crystal structure prediction pipeline, biomolecular structure refinement against experimental datasets, and estimation of the thermodynamic effects of genetic variants on both proteins and nucleic acids. The use of Parallel Java and OpenMM combines to offer shared memory, message passing, and graphics processing unit parallelization for high performance simulations. Overall, the FFX platform serves as a computational microscope to study systems ranging from organic crystals to solvated biomolecular systems.

Poissonian Cellular Potts Models Reveal Nonequilibrium Kinetics of Cell Sorting.

Cellular Potts models are broadly applied across developmental biology and cancer research. We overcome limitations of the traditional approach, which reinterprets a modified Metropolis sampling as ad hoc dynamics, by introducing a physical timescale through Poissonian kinetics and by applying principles of stochastic thermodynamics to separate thermal and relaxation effects from athermal noise and nonconservative forces. Our method accurately describes cell-sorting dynamics in mouse-embryo development and identifies the distinct contributions of nonequilibrium processes, e.g., cell growth and active fluctuations.

Thermodynamic profiles for cotranslational trigger factor substrate recognition.

Molecular chaperones are central to the maintenance of proteostasis in living cells. A key member of this protein family is trigger factor (TF), which acts throughout the protein life cycle and has a ubiquitous role as the first chaperone encountered by proteins during synthesis. However, our understanding of how TF achieves favorable interactions with such a diverse substrate base remains limited. Here, we use microfluidics to reveal the thermodynamic determinants of this process. We find that TF binding to empty 70S ribosomes is enthalpy-driven, with micromolar affinity, while nanomolar affinity is achieved through a favorable entropic contribution for both intrinsically disordered and folding-competent nascent chains. These findings suggest a general mechanism for cotranslational TF function, which relies on occupation of the exposed TF-substrate binding groove rather than specific complementarity between chaperone and nascent chain. These insights add to our wider understanding of how proteins can achieve broad substrate specificity.

Effective removal of chromium by adsorption using Delonix regia bark derived activated carbon from aqueous solution: a sustainable approach.

This study introduces a new biosorbent derived from Delonix regia bark-activated carbon to efficiently remove Chromium Cr(VI) metal ions from aqueous systems. The biosorbent was synthesized from the bark powder of the plant species and chemically activated with phosphoric acid. The biosorbent was characterized using FTIR, SEM, and BET to determine its functional properties and structural morphology. The batch adsorption experiments examined the optimal conditions for Cr(VI) metal ion adsorption, identifying that the highest removal efficiency occurred at pH levels of 2. The ideal adsorbent dosage was determined to be 2.5 g/L, with equilibrium achieved at a contact time of 60 min at the optimal temperature of about 303 K for a Cr(VI) metal ion concentration of 20 mg/L. Various isotherm models were applied to the adsorption equilibrium values, revealing that the adsorbent had a maximum removal capacity of approximately 224.8 mg/g for Cr(VI) metal ions. The adsorption process of Cr(VI) on the DAC biosorbent was best described by the Freundlich isotherm, indicating multilayer adsorption. The kinetic data fit well with the pseudo-second-order model. Thermodynamic parameters suggested that the adsorption process was spontaneous, exothermic, and feasible across different temperatures. Furthermore, the desorption studies showed that the DAC biosorbent can easily be rejuvenated and utilized several cycles with high adsorption capacity. These findings indicate that the developed adsorbent is environmentally friendly and effective for removing Cr(VI) from water systems.

Software for Predicting Binding Free Energy of Protein-Protein Complexes and Their Mutants.

Protein-protein binding affinity prediction is important for understanding complex biochemical pathways and to uncover protein interaction networks. Quantitative estimation of the binding affinity changes caused by mutations can provide critical information for protein function annotation and genetic disease diagnoses. The binding free energies of protein-protein complexes can be predicted using several computational tools. This chapter is a summary of software developed for the prediction of binding free energies for protein-protein complexes and their mutants.

Exploring Ficus religiosa inflorescence powder as an eco-friendly and sustainable solution for the removal of crystal violet with a disposal solution.

This study investigates the potential of using Ficus religiosa inflorescence (peepal tree) as an efficient solution for removing crystal violet from simulated and industrial wastewater. Various analyses were conducted to understand the adsorbent's structure, including particle morphology, BET surface area, FTIR, and pHZPC. The adsorption process was studied under different physicochemical factors such as temperature, concentration, contact time, and pH. Results revealed rapid adsorption, with 94.15% removal efficiency within the first 15 min at neutral pH. The highest observed adsorption capacity was 198.03 mg g-1, following pseudo-second-order kinetics (R2 = 0.99), indicating chemisorption. The Langmuir model accurately described the adsorption pathway (R2 = 0.99), showing monolayer adsorption. Thermodynamic analysis indicated an exothermic, feasible, and spontaneous process with increased entropy. The adsorbent could be easily regenerated using a 1:1 MeOH/H2O mixture for up to three cycles, yielding up to 73.86%. Real-time application with industrial effluent containing crystal violet showed up to 44.70% adsorption. The experiments demonstrated reliability with evaluated standard deviations (0.017935-0.000577) and relative standard deviations (0.439-0.673%), confirming statistical reliability. In conclusion, it presents a sustainable and eco-friendly approach for removing crystal violet dye from diverse wastewater sources.

NMR Dynamic View of the Destabilization of WW4 Domain by Chaotropic GdmCl and NaSCN.

GdmCl and NaSCN are two strong chaotropic salts commonly used in protein folding and stability studies, but their microscopic mechanisms remain enigmatic. Here, by CD and NMR, we investigated their effects on conformations, stability, binding and backbone dynamics on ps-ns and µs-ms time scales of a 39-residue but well-folded WW4 domain at salt concentrations ≤200 mM. Up to 200 mM, both denaturants did not alter the tertiary packing of WW4, but GdmCl exerted more severe destabilization than NaSCN. Intriguingly, GdmCl had only weak binding to amide protons, while NaSCN showed extensive binding to both hydrophobic side chains and amide protons. Neither denaturant significantly affected the overall ps-ns backbone dynamics, but they distinctively altered µs-ms backbone dynamics. This study unveils that GdmCl and NaSCN destabilize a protein before the global unfolding occurs with differential binding properties and µs-ms backbone dynamics, implying the absence of a simple correlation between thermodynamic stability and backbone dynamics of WW4 at both ps-ns and µs-ms time scales.

Comprehensive Investigation of [Fe(EDTA)]--Functionalized Derivatives and their Supramolecular Adducts with Human Serum Albumin.

In recent years, the coordination chemistry of high-spin Fe(III) complexes has increasingly attracted interest due to their potential as effective alternatives to Gd(III)-based MRI contrast agents. This paper discusses the results from our study on Fe(III) complexes with two EDTA derivatives, each modified with either one (EDTA-BOM) or two (EDTA-BOM2) benzyloxymethylene (BOM) groups on the acetic arm(s). These pendant hydrophobic groups enable the complexes to form noncovalent adducts with human serum albumin (HSA), leading to an observed increase in relaxivity due to the reduction in molecular tumbling. Our research involved detailed relaxometric measurements and analyses of both 1H and 17O NMR data at varying temperatures and magnetic field strengths, which is conducted with and without the presence of a protein. A significant finding of this study is the effect of electronic relaxation time on the effectiveness of [Fe(EDTA-BOM)(H2O)]- and [Fe(EDTA-BOM2)(H2O)]- as diagnostic MRI probes. By integrating these relaxometric results with comprehensive thermodynamic, kinetic, and electrochemical data, we have thoroughly characterized how structural modifications to the EDTA base ligand influence the properties of the complexes.

HSPiP, Computational, and Thermodynamic Model-Based Optimized Solvents for Subcutaneous Delivery of Tolterodine Tartrate and GastroPlus­Based In Vivo Prediction in Humans: Part II.

In part I, we reported Hansen solubility parameters (HSP, HSPiP program), experimental solubility at varied temperatures for TOTA delivery. Here, we studied dose volume selection, stability, pH, osmolality, dispersion, clarity, and viscosity of the explored combinations (I-VI). Ex vivo permeation and deposition studies were performed to observe relative diffusion rate from the injected site in rat skin. Confocal laser scanning microscopy (CLSM) study was conducted to support ex vivo findings. Moreover, GastroPlus predicted in vivo parameters in humans and the impact of various critical factors on pharmacokinetic parameters (PK). Immediate release product (IR) contained 60% of PEG400 whereas controlled release formulation (CR) contained PEG400 (60%), water (10%) and d-limonene (30%) to deliver 2 mg of TOTA. GastroPlus predicted the plasma drug concentration of weakly basic TOTA as function of pH (from pH 2.0 to 9). The cumulative drug permeation and drug deposition were found to be in the order as B-VI˃ C-VI˃A-VI across rat skin. This finding was further supported with CLSM. Moreover, IR and CR were predicted to achieve Cmax of 0.0038 µg/ mL and 0.00023 µg/mL, respectively, after sub-Q delivery. Added limonene in CR extended the plasma drug concentration over period of 12 h as predicted in GastroPlus. Parameters sensitivity analysis (PSA) assessment predicted that sub-Q blood flow rate is the only factor affecting PK parameters in IR formulation whereas this was insignificant for CR. Thus, sub-Q delivery CR would be promising alternative with ease of delivery to children and aged patient.

Binary bilayer simulations for partitioning within membranes.

Membrane proteins (MPs) often show preference for one phase over the other, which is characterized by the partition coefficient, Kp. The physical mechanisms underlying Kp have been only inferred indirectly from experiments due to the unavailability of detailed structures and compositions of ordered phases. Molecular dynamics (MD) simulations can complement these details and thus, in principle, provide further insights into the partitioning of MPs between two phases. However, the application of MD has remained difficult due to long time scales required for equilibration and large system size for the phase stability, which have not been fully resolved even in free energy simulations. This chapter describes the recently developed binary bilayer simulation method, where the membrane is composed of two laterally attached membrane patches. The binary bilayer system (BBS) is designed to preserve the lateral packing of both phases in a significantly smaller size compared to that required for macroscopic phase separation. These characteristics are advantageous in partitioning simulations, as the length scale for diffusion across the system can be significantly smaller. Hence the BBS can be efficiently employed in both conventional MD and free energy simulations, though sampling in ordered phases remains difficult due to slow diffusion. Development of efficient lipid swapping methods and its combination with the BBS would be a useful approach for partitioning in coexisting phases.

Modeling the mechanochemical feedback for membrane-protein interactions using a continuum mesh model.

The Helfrich free energy is widely used to model the generation of membrane curvature due to different physical and chemical components. The governing equations resulting from the energy minimization procedure are a system of coupled higher order partial differential equations. Simulations of membrane deformation for obtaining quantitative comparisons against experimental observations require computational schemes that will allow us to solve these equations without restrictions to axisymmetric coordinates. Here, we describe one such tool that we developed in our group based on discrete differential geometry to solve these equations along with examples.

Molecular insights into the interactions between PEG carriers and drug molecules from Celastrus hindsii: a multi-scale simulation study.

Efficient drug delivery is crucial for the creation of effective pharmaceutical treatments, and polyethylene glycol (PEG) carriers have been emerged as promising candidates for this purpose due to their bio-compatibility, enhancement of drug solubility, and stability. In this study, we utilized molecular simulations to examine the interactions between PEG carriers and selected drug molecules extracted from Celastrus hindsii: Hindsiilactone A, Hindsiiquinoflavan B, Maytenfolone A, and Celasdin B. The simulations provided detailed insights into the binding affinity, stability, and structural properties of these drug molecules when complexed with PEG carriers. A multi-scale approach combining density functional theory (DFT), extended tight-binding (xTB), and molecular dynamics (MD) simulations was conducted to investigate both unbound and bound states of PEG/drug systems. The results from DFT and xTB calculations revealed that the unbound complex has an unfavorable binding free energy, primarily due to negative contributions of delta solvation free energy and entropy. The MD simulations provided more detailed insights into the interactions between PEG and drug molecules in water solutions. By integrating the findings from the multi-scale simulations, a comprehensive picture of the unbound and bound states of PEG and drug systems were obtained. This information is valuable for understanding the molecular mechanisms governing the binding of drugs in PEG-based delivery platforms, and it contributes to the rational design and optimization of these systems.

Synthesis, application and molecular docking of modified cellulose with diaminoguanidine as complexing agent for selective separation of Cu (II), Cd (II) and Hg (II) ions from alum sample.

A new modified cellulose with diaminoguanidine (Cel-Gua) synthesized for specific recovery of Cu (II), Cd (II), and Hg (II) from the alum sample. Cellulose was silanized by 3-chloropropyltrimethoxysilane and then was modified with diaminoguanidine to obtain N-donor chelating fibers. Fourier transform-infrared spectroscopy, scanning electron microscopy, X-ray diffraction, zeta potential, electrons disperse X-ray analysis, elemental analyses (C, H and N), and thermogravimetric analysis were used for characterization. Factors influencing the adsorption were thoroughly examined. Under the optimal conditions, the Cel-Gua sorbent displayed maximum adsorption capacities of 94.33, 112.10 and 95.78 mg/g for Cu (II), Cd (II), and Hg (II), respectively. The sorption process of metal ions is equipped by kinetic model PSO and Langmuir adsorption isotherm. The calculated thermodynamic variables confirmed that the adsorption of Cu (II), Cd (II) and Hg (II) by Cel-Gua sorbent is a spontaneous and exothermic process. In our study, we used the molecular operating environment software to conduct molecular docking simulations on the Cel-Gua compound. The results of the docking simulations showed that the Cel-Gua compound displayed greater potency and a stronger affinity for the Avr2 effector protein derived from Fusarium oxysporum, a fungal plant pathogen (code 5OD4). The adsorbent was stable for 7 cycles, thus allowing its safe reutilization.

The mechanism of mammalian proton-coupled peptide transporters.

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

The cells in our body are sealed by a surrounding membrane that allows them to control which molecules can enter or leave. Desired molecules are often imported via transport proteins that require a source of energy. One way that transporter proteins achieve this is by simultaneously moving positively charged particles called protons across the membrane. Proteins called POTs (short for proton-coupled oligopeptide transporters) use this mechanism to import small peptides and drugsin to the cells of the kidney and small intestine. Sitting in the centre of these transporters is a pocket that binds to the imported peptide which has a gate on either side: an outer gate that opens towards the outside of the cell, and an inner gate that opens towards the cell's interior. The movement of protons from the outer to the inner gate is thought to shift the shape of the transporter from an outwards to an inwards-facing state. However, the molecular details of this energetic coupling are not well understood. To explore this, Lichtinger et al. used computer simulations to pinpoint where protons bind on POTs to trigger the gates to open. The simulations proposed that two sites together make up the outward-facing gate, which opens upon proton binding. Lichtinger et al. then validated these sites experimentally in cultured human cells that produce mutant POTs. After the desired peptide/drug has attached to the binding pocket, the protons then move to two more sites further down the transporter. This triggers the inner gate to open, which ultimately allows the small molecule to move into the cell. These findings represent a significant step towards understanding how POTs transport their cargo. Since POTs can transport a range of drugs from the digestive tract into the body, these results could help researchers design molecules that are better absorbed. This could lead to more orally available medications, making it easier for patients to adhere to their treatment regimen.

Stability Oracle: a structure-based graph-transformer framework for identifying stabilizing mutations.

Engineering stabilized proteins is a fundamental challenge in the development of industrial and pharmaceutical biotechnologies. We present Stability Oracle: a structure-based graph-transformer framework that achieves SOTA performance on accurately identifying thermodynamically stabilizing mutations. Our framework introduces several innovations to overcome well-known challenges in data scarcity and bias, generalization, and computation time, such as: Thermodynamic Permutations for data augmentation, structural amino acid embeddings to model a mutation with a single structure, a protein structure-specific attention-bias mechanism that makes transformers a viable alternative to graph neural networks. We provide training/test splits that mitigate data leakage and ensure proper model evaluation. Furthermore, to examine our data engineering contributions, we fine-tune ESM2 representations (Prostata-IFML) and achieve SOTA for sequence-based models. Notably, Stability Oracle outperforms Prostata-IFML even though it was pretrained on 2000X less proteins and has 548X less parameters. Our framework establishes a path for fine-tuning structure-based transformers to virtually any phenotype, a necessary task for accelerating the development of protein-based biotechnologies.

Electricity-driven dealkalization of bauxite residue based on thermodynamics, kinetics, and mineral transformation.

High alkalinity content of bauxite residue is a major factor that hinders resource reutilization and pollutes the environment. Although acid neutralization is a direct and effective method, the amount of acid and secondary waste of sodium salt are still difficult problems to solve. Herein, we innovatively integrated an electric field into the acid neutralization dealkalization of bauxite residue and analyzed the dealkalization behavior by thermodynamics, kinetics, and mineral transformation. The results show that the pH of the anode chamber was maintained at the acidic levels of 3-6 after 30 min of galvanostatic electrolysis, and bauxite residue can realize dealkalization by acid neutralization. In the anode chamber, Na+ was released into the leachate via the reactions of Na3Al3Si3O12 and the removal of encapsulated soluble alkali. The stainless steel wire mesh anode exhibited its superiority and decreased the Na2O content in bauxite residue from 9.48 to 3.13% through convective mass transfer driven by the electric field and steady-state diffusion under stirring. This research provides a promising method for the electricity-driven dealkalization of bauxite residue, thus facilitating the development of multifield coupling theory and the application of electric fields in the alumina industry.

Proton Transfer via Arginine with Suppressed pKa Mediates Catalysis by Gentisate and Salicylate Dioxygenase.

Gentisate and salicylate 1,2-dioxygenases (GDO and SDO) facilitate aerobic degradation of aromatic rings by inserting both atoms of dioxygen into their substrates, thereby participating in global carbon cycling. The role of acid-base catalysts in the reaction cycles of these enzymes is debatable. We present evidence of the participation of a proton shuffler during catalysis by GDO and SDO. The pH dependence of Michaelis-Menten parameters demonstrates that a single proton transfer is mandatory for the catalysis. Measurements at variable temperatures and pHs were used to determine the standard enthalpy of ionization (ΔHion°) of 51 kJ/mol for the proton transfer event. Although the observed apparent pKa in the range of 6.0-7.0 for substrates of both enzymes is highly suggestive of a histidine residue, ΔHion° establishes an arginine residue as the likely proton source, providing phylogenetic relevance for this strictly conserved residue in the GDO family. We propose that the atypical 3-histidine ferrous binding scaffold of GDOs contributes to the suppression of arginine pKa and provides support for this argument by employing a 2-histidine-1-carboxylate variant of the enzyme that exhibits elevated pKa. A reaction mechanism considering the role of the proton source in stabilizing key reaction intermediates is proposed.