Change In Net Charge Research Articles

Developing analytical ion exchange chromatography methods for antibody drug conjugates containing the hydrolysis-prone succinimide-thioether conjugation chemistry

Charge variants are one of the most important quality attributes for protein therapeutics, including antibody drug conjugates (ADCs). ADCs are conjugation products between monoclonal antibodies (mAbs) and highly potent payloads. After attaching a payload, the charge profile of a mAb can be modified due to the change in net charge or surface charge. In this study, we present a unique challenge of charge assay development that arises from a desirable engineering of ADCs that incorporates the hydrolysis-prone succinimide-thioether conjugation chemistry. This engineered hydrolysis at conjugation sites is usually not complete during conjugation process and continuously progressing during mild stress. This hydrolysis also creates a carboxylic functional group, which manifests as acidic peaks in the ADC charge profiles. As a result, ion exchange chromatograms become sensitive measurements of this hydrolysis, which often masks the charge profile change due to other important post-translational modifications. In this study, two approaches were explored to address this unique challenge: to remove the hydrolysis heterogeneity by incubating ADCs under high pH conditions to drive complete hydrolysis; and to analyze charge variants at the subunit level after IdeS digestion. Acceptable charge profiles and quantitative integration results were successfully obtained by both approaches.

Unique myoglobin adaptation to endothermy and flight since the origin of birds.

Myoglobin (Mb) mediates oxygen diffusion and storage in muscle tissue and thus is important for the energy utilization and activity of animals. Birds generally have a high body temperature, and most species also possess the capability of powered flight. Both of these require high levels of aerobic metabolism. Within endothermic mammals, bats also independently evolved flight. Although the functional evolution of myoglobins in deep-diving amniote vertebrates has been well-studied, the functional evolution of myoglobin since the origins of both birds and bats is unclear. Here, with Mb-coding sequences from >200 extant amniote species, we reconstructed ancestral sequences to estimate the functional properties of myoglobin through amniote evolution. A dramatic change in net surface charge on myoglobin occurred during the origin of Aves, which might have been driven by positively selected amino acid substitutions that occurred on the lineage leading to all birds. However, in bats, no change in net surface charge occurred and instead, the Mb genes show evidence of strong purifying selection. The increased net surface charge on bird myoglobins implies an adaptation to flight-related endothermic and higher body temperatures, possibly by reducing harmful protein aggregations. Different from the findings of net surface charge, myoglobins of extant birds show lower stability compared with other amniotes, which probably accelerates the rate of oxygen utilization in muscles. In bats and other mammals, higher stability of Mb may be an alternative pathway for adaptation to endothermy, indicating divergent evolution of myoglobin in birds and bats.

Massively parallel free energy calculations for in silico affinity maturation of designed miniproteins.

Computational protein design efforts continue to make remarkable advances, yet the discovery of high-affinity binders typically requires large-scale experimental screening of site-saturated mutant (SSM) libraries. Here, we explore how massively parallel free energy methods can be used for in silico affinity maturation of de novo designed binding proteins. Using an expanded ensemble (EE) approach, we perform exhaustive relative binding free energy calculations for SSM variants of three miniproteins designed to bind influenza A H1 hemagglutinin by Chevalier et al. (2017). We compare our predictions to experimental ΔΔ G values inferred from a Bayesian analysis of the high-throughput sequencing data, and to state-of-the-art predictions made using the Flex ddG Rosetta protocol. A systematic comparison reveals prediction accuracies around 2 kcal/mol, and identifies net charge changes, large numbers of alchemical atoms, and slow side chain conformational dynamics as key contributors to the uncertainty of the EE predictions. Flex ddG predictions are more accurate on average, but highly conservative. In contrast, EE predictions can better classify stabilizing and destabilizing mutations. We also explored the ability of SSM scans to rationalize known affinity-matured variants containing multiple mutations, which are non-additive due to epistatic effects. Simple electrostatic models fail to explain non-additivity, but observed mutations are found at positions with higher Shannon entropies. Overall, this work suggests that simulation-based free energy methods can provide predictive information for in silico affinity maturation of designed miniproteins, with many feasible improvements to the efficiency and accuracy within reach.

The Effect of the PhoP/PhoQ System on the Regulation of Multi-Stress Adaptation Induced by Acid Stress in Salmonella Typhimurium.

Acidic stress in beef cattle slaughtering abattoirs can induce the acid adaptation response of in-plant contaminated Salmonella. This may further lead to multiple resistance responses threatening public health. Therefore, the acid, heat, osmotic and antibiotic resistances of Salmonella typhimurium (ATCC14028) were evaluated after a 90 min adaption in a pH = 5.4 "mild acid" Luria-Bertani medium. Differences in such resistances were also determined between the ∆phoP mutant and wild-type Salmonella strains to confirm the contribution of the PhoP/PhoQ system. The transcriptomic differences between the acid-adapted and ∆phoP strain were compared to explore the role of the PhoP/Q two-component system in regulating multi-stress resistance. Acid adaptation was found to increase the viability of Salmonella to lethal acid, heat and hyperosmotic treatments. In particular, acid adaptation significantly increased the resistance of Salmonella typhimurium to Polymyxin B, and such resistance can last for 21 days when the adapted strain was stored in meat extract medium at 4 °C. Transcriptomics analysis revealed 178 up-regulated and 274 down-regulated genes in the ∆phoP strain. The Salmonella infection, cationic antimicrobial peptide (CAMP) resistance, quorum sensing and two-component system pathways were down-regulated, while the bacterial tricarboxylic acid cycle pathways were up-regulated. Transcriptomics and RT-qPCR analyses revealed that the deletion of the phoP gene resulted in the down-regulation of the expression of genes related to lipid A modification and efflux pumps. These changes in the gene expression result in the change in net negative charge and the mobility of the cell membrane, resulting in enhanced CAMP resistance. The confirmation of multiple stress resistance under acid adaptation and the transcriptomic study in the current study may provide valuable information for the control of multiple stress resistance and meat safety.

The Methods and Strategies to Improve the Stability of Nano Antibody

Because of the advantages of simple structure, easy modification, low immunogenicity, good stability, strong specificity and strong affinity, nano-antibodies have broad development space in clinical practice. Compared with traditional antibodies, nano antibodies are easier to store and transport, and after denaturation in high temperature, chemical, pressure and other environments, they can still be efficiently recombined, thereby regaining affinity for antigens. The structure and stability of nano antibody were studied. Multiple DNA sequence repair with the same sequence, easily modified amino acid replacement, protein net charge change, unnatural disulfide bond introduction and CDR supervariable region transfer was used to control the stability of the protein. The implementation of this project is expected to lay a foundation for improving its application in the fields of drug therapy, diagnosis and biosensing.

Edge Substitution Effects of Histidine Tautomerization Behaviors on the Structural Properties and Aggregation Properties of Aβ(1-42) Mature Fibril.

Histidine behaviors play critical roles in folding and misfolding processes due to the changes in net charge and the various N/N-H orientations on imidazole rings. However, the effect of histidine tautomerization (HIE (Nε-H, ε) and HID (Nδ-H, δ) states) behaviors on the edge chain of Aβ mature fibrils remains inadequately understood, which is critical for finding a strategy to disturb fibril elongation and growth. In the current study, eight independent molecular dynamics simulations were conducted to investigate such impacts on the structural and aggregation properties. Our results from three different binding models revealed that the binding contributions of edge substitution effects are primarily located between chains 1 and 2. Histidine states significantly influence the secondary structure of each domain. Further analysis confirmed that the C1_H6//C1_E11 intrachain interaction is essential in maintaining the internal stability of chain 1, while the C1_H13//C2_H13 and C1_H14//C2_H13 interchain interactions are critical in maintaining the interchain stability of the fibril structure. Our subsequent analysis revealed that the current edge substitution leads to the loss of the C1_H13//C1_E11 intrachain and C1_H13//C2_H14 interchain interactions. The N-terminal regularity was significantly directly influenced by histidine states, particularly by the residue of C1_H13. Our study provides valuable insights into the effect of histidine behaviors on the edge chain of Aβ mature fibril, advancing our understanding of the histidine behavior hypothesis in misfolding diseases.

Enhancement of functional properties, digestive properties, and in vitro digestion product physiological activity of extruded corn gluten meal by enzymatic modification.

Enzymatic modification is an effective means of improving the functional properties, digestive properties, and in vitro digestion product physiological activity of proteins, thus significantly expanding protein uses in various food applications. In this study, the addition of chymotrypsin (CT) at pH 9.0 and 11.0 was found to significantly improve the functional properties (solubility, foaming properties, water holding capacity, oil holding capacity, etc.) and digestive properties of extruded corn gluten meal (ECGM). Similar changes were observed when treating ECGM with glutaminase, protein glutaminase, and papain. These changes were likely due to the increase in number of carboxyl groups and the multiple effects of change in protein net charge and conformation caused by enzymatic deamidation. Of note, ECGM deamidated by CT showed the highest degree of deamidation, solubility, and gastrointestinal digestibility at pH 11.0, up to 44.92%, 43.75%, and 82.22%, respectively. In addition, CT-ECGM digestion product exhibited strong antioxidant activity and potential to promote alcohol metabolism in both a static digestion model and dynamic digestion model, even comparable to commercial corn peptides (CCP), while being inexpensive and of low bitterness compared to CCP. Meanwhile, the physiological activity enhanced as the molecular weight of digestion product decreased with the digested component having strongest activity. This study may promote the application of ECGM as a food component in the food industry or even as a substitute for CCP. © 2023 Society of Chemical Industry.

Guidelines for Free-Energy Calculations Involving Charge Changes.

The Coulomb interactions in molecular simulations are inherently approximated due to the finite size of the molecular box sizes amenable to current-day compute power. Several methods exist for treating long-range electrostatic interactions, yet these approaches are subject to various finite-size-related artifacts. Lattice-sum methods are frequently used to approximate long-range interactions; however, these approaches also suffer from artifacts which become particularly pronounced for free-energy calculations that involve charge changes. The artifacts, however, also affect the sampling when plain simulations are performed, leading to a biased ensemble. Here, we investigate two previously described model systems to determine if artifacts continue to play a role when overall neutral boxes are considered, in the context of both free-energy calculations and sampling. We find that ensuring that no net-charge changes take place, while maintaining a neutral simulation box, may be sufficient provided that the simulation boxes are large enough. Addition of salt to the solution (when appropriate) can further alleviate the remaining artifacts in the sampling or the calculated free-energy differences. We provide practical guidelines to avoid finite-size artifacts.

Proton-coupled electron transfer at a mis-metalated zinc site detected with protein charge ladders.

Distinguishing proton-coupled electron transfer (PCET) from uncoupled electron transfer (ET) in proteins can be challenging. A recent investigation [J. C. Koone, M. Simmang, D. L. Saenger, M. L. Hunsicker-Wang and B. F. Shaw, J. Am. Chem. Soc., 145, 16488-16497] reported that protein charge ladders and capillary electrophoresis can distinguish between single ET, PCET, and two-proton coupled ET (2PCET) by directly measuring the change in protein net charge upon reduction/oxidation (ΔZET). The current study used similar methods to assess PCET in zinc-free, "double copper" superoxide dismutase-1 (4Cu-SOD1), where one copper is bound at the copper site of each monomer and one copper is bound at the bridging zinc site, resulting in a quasi-type III Cu center. At pH 7.4, the net charge (Z) of the 4Cu-SOD1 dimer was unaffected by reduction of all four Cu2+ ions, i.e., ΔZ4ET = -0.09 ± 0.05 per dimer (-0.02 ± 0.01 per copper atom). These values suggest that PCET is taking place at all four Cu atoms of the homodimer. Molecular dynamics and Poisson-Boltzmann calculations suggest that a metal-coordinating histidine at the zinc site (His71) is the proton acceptor. These data show how ligands of a naturally occurring zinc site can help facilitate PCET when the right redox metal is bound.

New Hypothesis Insight of Structural Properties on Four Tau Peptide Fragments in Alzheimer's Disease: Origin from the Histidine Behaviors.

During protein folding and misfolding, structural properties and aggregation tendency can be significantly influenced by histidine behaviors (tautomeric behaviors and protonation behaviors). The original reasons were derived from the net charge changes and the various N/N-H orientation on imidazole rings. In the current study, total 18 independent REMD simulations were performed to investigate the histidine behaviors on four Tau peptide fragments (MBD, including R1, R2, R3, and R4 fragments). We found that, compared to R1, R2, R3 except (εδ), and R4 systems with flexible structural features, only R3(εδ) has dominating conformational structure (possibility of 81.3%) with three β-strand structures in parallel β-sheet structures at I4-K6and I24-H26, as well as antiparallel β-sheet structure at G19-L21. Importantly, the H25 and H26 residues (in R3(εδ) system) are directly involved in the sheet structure formations and strong H-bonded interactions (possibility range of 31.3%-44.7%). Furthermore, the donors and acceptors analysis confirmed that only R3(εδ) shows faraway amino acids interaction features in both H25 and H26 residues, and such cooperation effects of two histidine residues contribute to current structural features. The current study will be helpful to further enrichment of the histidine behavior hypothesis, it provides new insight for understanding protein folding and misfolding.

New Insights into the Structural and Binding Properties on Aβ Mature Fibrils Due to Histidine Protonation Behaviors.

Histidine tautomeric behaviors have been considered origin factors for controlling the structure and aggregation properties of misfolding peptides. Except for tautomeric behaviors, histidine protonation behaviors definitely have the same capacities due to the net charge changes and the various N/N-H orientations on imidazole rings. However, such phenomena are still unknown. In the current study, Aβ mature fibrils substituted with various protonation states were performed by molecular dynamics simulations to investigate the structure and binding properties. Our results show that all kinds of protonation states can increase the ΔG1 stability and decrease ΔG2 and ΔG3 stabilities. A significantly higher averaged β-sheet content was detected in (εεp), (εpp), and (ppp) fibrils in one, two, and three protonation stages, respectively. Impressively, we found that the substituted fibril with specific protonated states can control the N-terminus structural properties. Further analysis confirmed that H6 and H13 are more important than H14 since the H-bond donor and receptor cooperate among C1/C3/C8_H6, C1/C3/C8_H13, and C1/C3/C8_E11. Furthermore, the mechanism of protonation behaviors was discussed. The current study is helpful for understanding the histidine protonation behaviors on one, two, and three protonation stages, which provides new horizons for exploring the origin of protein folding and misfolding.

First principles study of hydrogen adsorption and dissociation behavior on γ-U (100)/Mo surface

<sec>Uranium (U) is one of the most natural radioactive elements widely used in the nuclear industry. In the civilian field, uranium is the most important nuclear fuel in nuclear reactors; militarily, uranium is an important raw material for nuclear weapons. In addition, uranium is also used for radiation shielding and ship ballast due to its high-density properties. Depending on the temperature, U has three kinds of allotrope phases: the orthogonal α phase at temperature below 940 K, the body-centered tetragonal (BCT) β phase at temperature ranging from 940 K to 1050 K, and the body-centered cubic (BCC) γ phase at temperature above 1050 K. Compared with the other two structures, the crystal structure of γ phase has good symmetry and excellent mechanical properties. However, γ-U is extremely unstable at low temperature. No matter what heat treatment method is adopted, γ-U will undergo phase transformation and become α phase. It is shown that adding certain alloying elements, such as Mo, Nb, Zr, Ti and Hf, into uranium can stabilize γ-U to room temperature and improve the mechanical properties of uranium greatly. As an important uranium alloy, γ-U by doping Mo atom has excellent mechanical properties, structural stability and thermal conductivity, and is an important nuclear reactor fuel.</sec><sec>However, uranium has special physical and chemical properties due to its complex electronic structure and strong correlation of 5f electrons. Because of its special valence electron structure, it is highly susceptible to chemical and electrochemical reactions of environmental media. After the reaction between uranium and hydrogen, hydrogen embrittlement will occur, and even easily break into powder, which reduces the performance of uranium in service and brings hidden trouble to its storage. With the increase of service life, surface corrosion becomes more serious, and the safety and reliability of U alloys are seriously affected. The adsorption and dissociation of hydrogen on U alloy surface is the primary process of hydrogenation corrosion.</sec><sec>Based on density functional theory, first-principles study of hydrogen adsorption and dissociation on γ-U(100) surface by Mo atoms coatings is carried out in this work. The model of γ-U(100) and Mo atoms coatings on γ-U(100) surface are established, and the structural parameters, adsorption energy, Bader charge, surface work function, and electron state density of H<sub>2</sub> at highly symmetrical adsorption sites are calculated. The results show that H<sub>2</sub> molecule occurs when physical dissociation adsorption takes place on γ-U(100) and U(100)/Mo surface. The electron state density shows that H<sub>2</sub> does not bond to the surface atoms and no new hybridization peak appears. However, in the hollow parallel adsorption configuration, H<sub>2</sub> is completely dissociated into two H atoms and occurs chemical adsorption and dissociation on γ-U(100) and U(100)/Mo surface. The H/1s orbital electrons are hybridized with the U/6p, U/6d, Mo/5s, Mo/4p, Mo/4d orbital electrons, and the H atom forms stable chemical bonds with the Mo atoms. Bader charge distribution results show that the change of chemical adsorption net charge of H<sub>2</sub> on U(100)/Mo is more than that of physical adsorption. Because the adsorption energy of H<sub>2</sub> in the most stable configuration (H<sub>Mo</sub>-Hor) on U(100)/Mo is less than that of the most stable configuration (H<sub>U</sub>-Hor) on γ-U(100), the adsorption of H<sub>2</sub> on U(100)/Mo is more stable than that of γ-U(100) surface.</sec>

Electrostatic-triggered exothermic antibody adsorption to the cellulose nanoparticles

Antibody-conjugated nanoparticles are used in a fields ranging from medicine to engineering. NanoAct® nanobeads are cellulose nanoparticles used in lateral flow assays that are highly water dispersible. In order to promote the adsorption of antibodies onto NanoAct® particles while maintaining their activity, we analyzed the adsorption onto NanoAct® particles thermodynamically and elucidated the adsorption mechanism. In an immunochromatographic assay, the amount of adsorbed antibody and the color intensity of the test line increased as the pH decreased. The zeta potential of the nanoparticles remained constant at around −30 mV over the pH range from 2 to 10. The model antibody had pI values between 6.2 and 6.8. Isothermal calorimetry analysis showed that adsorption of antibody to the NanoAct® particle is an endothermic reaction under low pH conditions, an exothermic reaction between pH 6 and pH 7, and a weakly exothermic reaction above pH 7. These data indicate that the changes in net charge of the antibody surface as a function of pH influence the pH dependence of antibody adsorption to the negatively charged NanoAct®. This suggests that increased positive charge on the antibody surface will result in a more sensitive NanoAct®-based immunoassay.

Characterization of Protein-Phospholipid/Membrane Interactions Using a "Membrane-on-a-Chip" Microfluidic System.

It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.

Charge-Changing Perturbations and Path Sampling via Classical Molecular Dynamic Simulations of Simple Guest-Host Systems.

Currently, two different methods dominate the field of biomolecular free-energy calculations for the prediction of binding affinities. Pathway methods are frequently used for large ligands that bind on the surface of a host, such as protein–protein complexes. Alchemical methods, on the other hand, are preferably applied for small ligands that bind to deeply buried binding sites. The latter methods are also widely known to be heavily artifacted by the representation of electrostatic energies in periodic simulation boxes, in particular, when net-charge changes are involved. Different methods have been described to deal with these artifacts, including postsimulation correction schemes and instantaneous correction schemes (e.g., co-alchemical perturbation of ions). Here, we use very simple test systems to show that instantaneous correction schemes with no change in the system net charge lower the artifacts but do not eliminate them. Furthermore, we show that free energies from pathway methods suffer from the same artifacts.

Implications of localized charge for human influenza A H1N1 hemagglutinin evolution: Insights from deep mutational scans.

Seasonal influenza A viruses of humans evolve rapidly due to strong selection pressures from host immune responses, principally on the hemagglutinin (HA) viral surface protein. Based on mouse transmission experiments, a proposed mechanism for immune evasion consists of increased avidity to host cellular receptors, mediated by electrostatic charge interactions with negatively charged cell surfaces. In support of this, the HA charge of the globally circulating H3N2 has increased over time since its pandemic. However, the same trend was not seen in H1N1 HA sequences. This is counter-intuitive, since immune escape due to increased avidity (due itself to an increase in charge) was determined experimentally. Here, we explore whether patterns of local charge of H1N1 HA can explain this discrepancy and thus further associate electrostatic charge with immune escape and viral evolutionary dynamics. Measures of site-wise functional selection and expected charge computed from deep mutational scan data on an early H1N1 HA yield a striking division of residues into three groups, separated by charge. We then explored evolutionary dynamics of these groups from 1918 to 2008. In particular, one group increases in net charge over time and consists of sites that are evolving the fastest, that are closest to the receptor binding site (RBS), and that are exposed to solvent (i.e., on the surface). By contrast, another group decreases in net charge and consists of sites that are further away from the RBS and evolving slower, but also exposed to solvent. The last group consists of those sites in the HA core, with no change in net charge and that evolve very slowly. Thus, there is a group of residues that follows the same trend as seen for the entire H3N2 HA. It is possible that the H1N1 HA is under other biophysical constraints that result in compensatory decreases in charge elsewhere on the protein. Our results implicate localized charge in HA interactions with host cells, and highlight how deep mutational scan data can inform evolutionary hypotheses.

Complete Charge Regulation by a Redox Enzyme Upon Single Electron Transfer

AbstractThe degree by which metalloproteins partially regulate net charge (Z) upon electron transfer (ET) was recently measured for the first time using “protein charge ladders” of azurin, cytochrome c, and myoglobin [Angew. Chem. Int. Ed. 2018, 57(19), 5364–5368; Angew. Chem. 2018, 130, 5462–5466]. Here, we show that Cu, Zn superoxide dismutase (SOD1) is unique among proteins in its ability to resist changes in net charge upon single ET (e.g., ΔZET(SOD1)=0.05±0.08 per electron, compared to ΔZET(Cyt‐c)=1.19±0.02). This total regulation of net charge by SOD1 is attributed to the protonation of the bridging histidine upon copper reduction, yielding redox centers that are isoelectric at both copper oxidation states. Charge regulation by SOD1 would prevent long range coulombic perturbations to residue pKa’s upon ET at copper, allowing SOD1’s “electrostatic loop” to attract superoxide with equal affinity (at both redox states of copper) during diffusion‐limited reduction and oxidation of superoxide.

Complete Charge Regulation by a Redox Enzyme Upon Single Electron Transfer.

The degree by which metalloproteins partially regulate net charge (Z) upon electron transfer (ET) was recently measured for the first time using "protein charge ladders" of azurin, cytochrome c, and myoglobin [Angew. Chem. Int. Ed. 2018, 57(19), 5364-5368; Angew. Chem. 2018, 130, 5462-5466]. Here, we show that Cu, Zn superoxide dismutase (SOD1) is unique among proteins in its ability to resist changes in net charge upon single ET (e.g., ΔZET(SOD1) =0.05±0.08 per electron, compared to ΔZET(Cyt-c) =1.19±0.02). This total regulation of net charge by SOD1 is attributed to the protonation of the bridging histidine upon copper reduction, yielding redox centers that are isoelectric at both copper oxidation states. Charge regulation by SOD1 would prevent long range coulombic perturbations to residue pKa 's upon ET at copper, allowing SOD1's "electrostatic loop" to attract superoxide with equal affinity (at both redox states of copper) during diffusion-limited reduction and oxidation of superoxide.

Correcting electrostatic artifacts due to net-charge changes in the calculation of ligand binding free energies.

Alchemically derived free energies are artifacted when the perturbed moiety has a nonzero net charge. The source of the artifacts lies in the effective treatment of the electrostatic interactions within and between the perturbed atoms and remaining (partial) charges in the simulated system. To treat the electrostatic interactions effectively, lattice-summation (LS) methods or cutoff schemes in combination with a reaction-field contribution are usually employed. Both methods render the charging component of the calculated free energies sensitive to essential parameters of the system like the cutoff radius or the box side lengths. Here, we discuss the results of three previously published studies of ligand binding. These studies presented estimates of binding free energies that were artifacted due to the charged nature of the ligands. We show that the size of the artifacts can be efficiently calculated and raw simulation data can be corrected. We compare the corrected results with experimental estimates and nonartifacted estimates from path-sampling methods. Although the employed correction scheme involves computationally demanding continuum-electrostatics calculations, we show that the correction estimate can be deduced from a small sample of configurations rather than from the entire ensemble. This observation makes the calculations of correction terms feasible for complex biological systems. To show the general applicability of the proposed procedure, we also present results where the correction scheme was used to correct independent free energies obtained from simulations employing a cutoff scheme or LS electrostatics. In this work, we give practical guidelines on how to apply the appropriate corrections easily.

The role of surface charge and pH changes in tropical soils on sorption behaviour of per- and polyfluoroalkyl substances (PFASs)

This study investigated the effect of surface charge on the sorption of perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA) and perfluorohexane sulfonic acid (PFHxS) onto 7 tropical soils as a function of pH. The net surface charge became less negative with decreasing pH (from 7.5 to 3.5) in all soils. The rate of change in net surface charge varied from −0.6 to −2.8 (cmol/kg)/pH unit. The effect on sorption behaviour of PFASs was variable among soils. For two soils, the average sorption increased 54- and 45-fold for PFOS, 33- and 9-fold for PFOA, and 39- and 400-fold for PFHxS, across the pH range 7.5 to 3.5. Sorption in another sandier soil showed negligible change with decreasing pH. Sorption in the other soils did not change significantly until the pH decreased to approximately 5.5. The soils with high contents of sesquioxides (Fe and Al oxides) showed the most marked increase in sorption with decreasing pH. This study demonstrated that in addition to hydrophobic interactions with OC and other processes, electrostatic interactions are also important in the sorption process for these chemicals in soils. In acidic, variably charged tropical soils there is the possibility that any PFOS, PFOA or PFHxS sorbed to the soils may become desorbed if management practices (e.g. liming) raised soil pH.