Protein Charge Distribution Research Articles

Improving Downstream Process Related Manufacturability Based on Protein Engineering—A Feasibility Study

ABSTRACTWhile bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process‐related manufacturability, that is, how easily can a protein be purified. Process‐related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety. Therefore, during molecule design, the surface charge of the target molecule should preferably differ sufficiently from the surface charge of the impurities to enable an efficient purification strategy. In this feasibility study, we evaluated the possibility of improving manufacturability by adapting the surface charge of the target protein. We generated several variants of a GLP1‐receptor‐agonist‐Fc‐domain‐FGF21‐fusion protein and demonstrated proof of concept exemplarily for an anion exchange chromatography step which then can be operated at high pH values with maximal product recovery allowing removal of HCP and viruses. Altering the surface charge distribution of biotherapeutic proteins can thus be useful allowing for an efficient manufacturing process for removing HCP and viruses, thereby reducing manufacturing costs.

Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria.

Subcellular phase-separated compartments, known as biomolecular condensates, play an important role in the spatiotemporal organization of cells. To understand the sequence-determinants of phase separation in bacteria, we engineered protein-based condensates in Escherichia coli using electrostatic interactions as the main driving force. Minimal cationic disordered peptides were used to supercharge negative, neutral, and positive globular model proteins, enabling their phase separation with anionic biomacromolecules in the cell. The phase behavior was governed by the interaction strength between the cationic proteins and anionic biopolymers, in addition to the protein concentration. The interaction strength primarily depended on the overall net charge of the protein, but the distribution of charge between the globular and disordered domains also had an impact. Notably, the protein charge distribution between domains could tune mesoscale attributes such as the size, number, and subcellular localization of condensates within E. coli cells. The length and charge density of the disordered peptides had significant effects on protein expression levels, ultimately influencing the formation of condensates. Taken together, charge-patterned disordered peptides provide a platform for understanding the molecular grammar underlying phase separation in bacteria.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.

Tau and tubulin protein aggregation characterization by solid-state nanopore method and atomic force microscopy

In this study, a silicon nitride nanopore-based sensing system was used to measure tau and tubulin monomers and their aggregations in salt solution at a single molecule level. Nanopores (6–30 nm) were fabricated on silicon nitride membranes supported by silicon substrates using a combination of focused ion beam milling and ion beam sculpting. When a charged protein molecule in the salt solution passes through a nanopore driven by an applied voltage, the protein molecule increases pore resistivity, which induces an ionic current drop that can be measured. The current drop amplitude is directly proportional to the local excluded volume of the protein molecule in the nanopore. We measured the monomers and aggregations of tau and tubulin proteins at biased voltages from 60 to 210 mV in a solution of pH 7.0–10. Our results showed that (1) the nanopore method was able to differentiate tau and tubulin proteins in their monomer and aggregated forms by their excluded volumes; (2) the most probable aggregation form was dimer for α- and β-tubulin and pentamer for αβ tubulin plus tau under experimental conditions; (3) the protein excluded volumes measured by the nanopore method depended on the applied voltage, and this observation could be explained by the nonuniform charge distribution of proteins. The monomer and aggregated proteins were further analyzed using atomic force spectroscopy (AFM), and protein volumes estimated by AFM were consistent with nanopore results.

Bioinspired Polyethylene Glycol Coatings for Reduced Nanoparticle-Protein Interactions.

Nanoparticles (NPs) and other engineered nanomaterials have great potential as nanodrugs or nanomedical devices for biomedical applications. However, the adsorption of proteins in blood circulation or similar physiological fluids can significantly alter the surface properties and therapeutic response induced by most nanomaterials. For example, interaction with proteins can change the bloodstream circulation time and availability of therapeutic NPs or hinder the accumulation in their desired target organs. Proteins can also trigger or prevent agglomeration. By combining experimental and computational approaches, we have developed NPs carrying polyethylene glycol (PEG) polymeric coatings that mimic the surface charge distribution of proteins typically found in blood, which are known to show low aggregation under normal blood conditions. Here, we show that NPs with coatings based on apoferritin or human serum albumin display better antifouling properties and weaker protein interaction compared to similar NPs carrying conventional PEG polymeric coatings.

Hidden symmetry of the flavivirus protein shell and pH-controlled reconstruction of the viral surface.

Using recent Zika virus structural data we reveal a hidden symmetry of protein order in immature and mature flavivirus shells, violating the Caspar-Klug paradigmatic model of capsid structures. We show that proteins of the outer immature shell layer exhibit trihexagonal tiling, while proteins from inner and outer layers conjointly form a double-shelled close-packed structure, based on a common triangular spherical lattice. Within the proposed structural model, we furthermore rationalize the structural organization of misassembled non-infectious subviral particles that have no inner capsid. We consider a pH-controlled structural reconstruction of the outer shell from the trimeric to the dimeric state, and demonstrate that this transition, occurring during the virus maturation, can be induced by changes in protein charges at lower pH, leading to a decrease in the electrostatic interaction free energy. This transition could also be assisted by electrostatic attraction of shell proteins to the interposed lipid membrane substrate separating the shells.

Impact of Charge Composition and Distribution on the Antibacterial Properties of Polypeptide Coatings.

Inspired by the charge composition and distribution of proteins and peptides, we designed and prepared a series of brush polypeptides with positive and negative charges separately distributed in the side chains and the backbones. The brush polypeptides can self- or co-deposit on various substrates forming ultrathin and stable coatings. They showed potent bactericidal activity and antibiofilm property, outperforming conventional linear polypeptide coatings with randomly distributed positive and negative charges. Keeping the balance of positive/negative charges and increasing the numbers of positive/negative charges can further improve the antibacterial property of brush polypeptide coatings without sacrificing their biocompatibility.

PatcHwork: a user-friendly pH sensitivity analysis web server for protein sequences and structures.

pH regulates protein function and interactions by altering the charge of individual residues causing loss or gain of intramolecular noncovalent bonds, which may lead to structural rearrangements. While tools to analyze residue-specific charge distribution of proteins at a given pH exist, currently no tool is available to investigate noncovalent bond changes at two different pH values. To make protein pH sensitivity analysis more accessible, we developed patcHwork, a web server that combines the identification of amino acids undergoing a charge shift with the determination of affected noncovalent bonds at two user-defined pH values. At the sequence-only level, patcHwork applies the Henderson–Hasselbalch equation to determine pH-sensitive residues. When the 3D protein structure is available, patcHwork can be employed to gain mechanistic understanding of the effect of pH. This is achieved using the PDB2PQR and PROPKA tools and noncovalent bond determination algorithms. A user-friendly interface allows visualizing pH-sensitive residues, affected salt bridges, hydrogen bonds and aromatic (pi–pi and cation–pi) interactions. patcHwork can be used to identify patches, a new concept we propose of pH-sensitive residues in close proximity on the protein, which may have a major impact on function. We demonstrate the attractiveness of patcHwork studying experimentally investigated pH-sensitive proteins (https://patchwork.biologie.uni-freiburg.de/).

Approach to Study pH-Dependent Protein Association Using Constant-pH Molecular Dynamics: Application to the Dimerization of β-Lactoglobulin.

Protein-protein association is often mediated by electrostatic interactions and modulated by pH. However, experimental and computational studies have often overlooked the effect of association on the protonation state of the protein. In this work, we present a methodological approach based on constant-pH molecular dynamics (MD), which aims to provide a detailed description of a pH-dependent protein-protein association, and apply it to the dimerization of β-lactoglobulin (BLG). A selection of analyses is performed using the data generated by constant-pH MD simulations of monomeric and dimeric forms of bovine BLG, in the pH range 3-8. First, we estimate free energies of dimerization using a computationally inexpensive approach based on the Wyman-Tanford linkage theory, calculated in a new way through the use of thermodynamically based splines. The individual free energy contribution of each titratable site is also calculated, allowing for identification of relevant residues. Second, the correlations between the proton occupancies of pairs of sites are calculated (using the Pearson coefficient), and extensive networks of correlated sites are observed at acidic pH values, sometimes involving distant pairs. In general, strongly correlated sites are also slow proton exchangers and contribute significantly to the pH-dependency of the dimerization free energy. Third, we use ionic density as a fingerprint of protein charge distribution and observe electrostatic complementarity between the monomer faces that form the dimer interface, more markedly at the isoionic point (where maximum dimerization occurs) than at other pH values, which might contribute to guide the association. Finally, the pH-dependent dimerization modes are inspected using PCA, among other analyses, and two states are identified: a relaxed state at pH 4-8 (with the typical alignment of the crystallographic structure) and a compact state at pH 3-4 (with a tighter association and rotated alignment). This work shows that an approach based on constant-pH MD simulations can produce rich detailed pictures of pH-dependent protein associations, as illustrated for BLG dimerization.

Surface charge distribution: a key parameter for understanding protein behavior in chromatographic processes

Multi-component adsorption of proteins still requires a better understanding of local phenomena to improve the development of predictive models. In this work, all-atom Molecular Dynamics (MD) simulations were used to investigate the influence of protein charge distribution on the adsorption capacity. The simultaneous adsorption of α-chymotrypsin and lysozyme on a cation exchanger, SP Sepharose FF, was studied through MD simulations and compared to macroscopic isotherm experiments. It appears that the charge distribution is a relevant information to better understand specific phenomena, such as a multilayer adsorption caused by the particular electrostatic profile of α-chymotrypsin. Therefore, MD simulations seem to be an interesting way to visualize and highlight these behaviors.

De novo sequence redesign of a functional Ras-binding domain globally inverted the surface charge distribution and led to extreme thermostability.

To acquire extremely thermostable proteins of given functions is challenging for conventional protein engineering. Here we applied ABACUS, a statistical energy function we developed for de novo amino acid sequence design, to globally redesign a Ras-binding domain (RBD), and obtained an extremely thermostable RBD that unfolds reversibly at above 110°C, the redesigned RBD experimentally confirmed to have expected structure and Ras-binding interface. Directed evolution of the redesigned RBD improved its Ras-binding affinity to the native protein level without excessive loss of thermostability. The designed amino acid substitutions were mostly at the protein surface. For many substitutions, strong epistasis or significantly differentiated effects on thermostability in the native sequence context relative to the redesigned sequence context were observed, suggesting the globally redesigned sequence to be unreachable through combining beneficial mutations of the native sequence. Further analyses revealed that by replacing 38 of a total of 48 non-interfacial surface residues at once, ABACUS redesign was able to globally "invert" the protein's charge distribution pattern in an optimized way. Our study demonstrates that computational protein design provides powerful new tools to solve challenging protein engineering problems.

Effect of the charge distribution of virus coat proteins on the length of packaged RNAs.

Single-stranded RNA viruses efficiently encapsulate their genome into a protein shell called the capsid. Electrostatic interactions between the positive charges in the capsid protein's N-terminal tail and the negatively charged genome have been postulated as the main driving force for virus assembly. Recent experimental results indicate that the N-terminal tail with the same number of charges and same lengths packages different amounts of RNA, which reveals that electrostatics alone cannot explain all the observed outcomes of the RNA self-assembly experiments. Using a mean-field theory, we show that the combined effect of genome configurational entropy and electrostatics can explain to some extent the amount of packaged RNA with mutant proteins where the location and number of charges on the tails are altered. Understanding the factors contributing to the virus assembly could promote the attempt to block viral infections or to build capsids for gene therapy applications.

Hypothesis: Potentially Systemic Impacts of Elevated CO2 on the Human Proteome and Health.

Uniform CO2 during human evolution (180 to 280 ppm) resulted, because of the role of the CO2-bicarbonate buffer in regulating pH, in rather constant pH (7.35 to 7.45) in human fluids, cells and tissues, determining, in turn, the narrow pH range for optimal functioning of the human proteome. Herein, we hypothesize that chronic exposure to elevated pCO2 with increasing atmospheric CO2 (>400 ppm), and extended time spent in confined, crowded indoor atmospheres (pCO2 up to 5,000 ppm) with urban lifestyles, may be an important, largely overlooked driver of change in human proteome performance. The reduced pH (downregulated from 0.1 to 0.4 units below the optimum pH) of extant humans chronically exposed to elevated CO2 is likely to lead to proteome malfunction. This malfunction is due to protein misfolding, aggregation, charge distribution, and altered interaction with other molecules (e.g., nucleic acids, metals, proteins, and drugs). Such alterations would have systemic effects that help explain the prevalence of syndromes (obesity, diabetes, respiratory diseases, osteoporosis, cancer, and neurological disorders) characteristic of the modern lifestyle. Chronic exposure to elevated CO2 poses risks to human health that are too serious to be ignored and require testing with fit-for-purpose equipment and protocols along with indoor carbon capture technologies to bring CO2 levels down to approach levels (180–280 ppm) under which the human proteome evolved.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome c Peroxidase with an Additional, Distant Electrostatic Binding Site.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome c Peroxidase with an Additional, Distant Electrostatic Binding Site

AbstractElectrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.

Molecular Simulation Elaborating the Mechanism of 1\u03b2-Hydroxy Alantolactone Inhibiting Ubiquitin-Conjugating Enzyme UbcH5s

1β-hydroxy alantolactone, a sesquiterpene lactone, exhibits potent anti-inflammatory and anticancer activities. Recently, it has been found to target UbcH5s by covalently bonding with Cys85 specifically, but the exact molecular basis remains unclear. Here, we analyzed the structural specificity of the catalytic site of UbcH5s by comparing them with other E2 proteins. Molecular dynamics was performed to detect the structural stability of the catalytic site. Docking method was then used to predict conformations of ligand docked at the catalytic site of UbcH5s. The electrostatic surface and charge distribution of ligand and proteins were analyzed by quantitative calculation. Molecular dynamics was used to detect the stability of docking complexes of 1β-hydroxy alantolactone and UbcH5s, the covalently bonded intermediates and the products. The QM/MM methodology was used to calculate the free energy barrier of hydrogen transfer and formation of covalent bond between 15-position carbon of ligand and Cys85. Results revealed that the structure of the catalytic site is stable, and 1β-hydroxy alantolactone can dock at the catalytic site with correct conformation. Molecular dynamics further demonstrates that 1β-hydroxy alantolactone can steadily combine with UbcH5s. Intermediate and product of catalytic reaction are also certified to be stable. Besides, Asp112 and Asn114 function as anchors to fix ligand, ensuring it steadily docked at catalytic site to complete covalent reaction. More importantly, we have found that Cys85 of UbcH5c is more efficient to form a covalent bond with the ligand in comparison with UbcH5a and UbcH5b. Our results successfully explained the mechanism of 1β-hydroxy alantolactone covalently bonding with UbcH5s. Such molecular mechanism may provide a better insight into the molecular development or modification for ubiquitin-related drugs.

Effect of pulsed electric field on assembly structure of α-amylase and pectin electrostatic complexes

Pulsed electric field (PEF) could change the charge distribution of proteins and polysaccharides and affect their interactions and complexes aggregation, but those influences are not enough evaluated. Here, the effects of PEF on the complexes of α-amylase and pectin driven by electrostatic binding were studied. Changes in molecular conformation of α-amylase and assembly structure of α-amylase/pectin complexes were orderly assessed by fluorescence, FTIR, DSC, enzyme activities, particle size, ζ-potential, CLSM, and SEM. After PEF treatment (E~20 kV/cm, texp~1 ms, and 5 cycles), the intrinsic fluorescence of α-amylase was quenched, the content of β-sheet increased, enzyme activities lose almost 80%, and the denatured temperature increased. Ζeta-potential of α-amylase/pectin complexes did not change significantly, but the particle size rose gradually. The particle revolution of α-amylase/pectin complexes was recorded by Turbiscan, and the size growth model fit the Allometric function well. Finally, the complexes of α-amylase and pectin after PEF treatment tended to the branched, ring, or circles-like shape.

A positive-charged patch and stabilized hydrophobic core are essential for avirulence function of AvrPib in the rice blast fungus.

Fungal avirulence effectors, a key weapon utilized by pathogens to promote their infection, are recognized by immune receptors to boost host R gene-mediated resistance. Many avirulence effectors share sparse sequence homology to proteins with known functions, and their molecular and biochemical functions together with the evolutionary relationship among different members remain largely unknown. Here, the crystal structure of AvrPib, an avirulence effector from Magnaporthe oryzae, was determined and showed a high degree of similarity to the M.oryzae Avrs and ToxB (MAX) effectors. Compared with other MAX effectors, AvrPib has a distinct positive-charge patch formed by five positive-charged residues (K29, K30, R50, K52 and K70) on the surface. These five key residues were essential to avirulence function of AvrPib and affected its nuclear localization into host cells. Moreover, residues V39 and V58, which locate in the hydrophobic core of the structure, cause loss of function of AvrPib by single-point mutation in natural isolates. In comparison with the wild-type AvrPib, the V39A or V58A mutations resulted in a partial or entire loss of secondary structure elements. Taken together, our results suggest that differences in the surface charge distribution of avirulence proteins could be one of the major bases for the variation in effector-receptor specificity, and that destabilization of the hydrophobic core is one of the major mechanisms employed by AvrPib for the fungus to evade recognition by resistance factors in the host cell.

The impact of the modified Poisson–Boltzmann model on protein bound to a lipid coated silicon nanowire field effect transistor biosensor in an electrolyte environment

ABSTRACTThe aim of this work was to analyse the electrostatic potential profile, various effects of electrolyte concentrations, and the influences of surface charge on a protein bound to a lipid coated Silicon nanowire field effect transistor (Si-NW FET) biosensor by implementing the modified Poisson–Boltzmann (MPB) model. In this work, we modelled a lipid monolayer-coated Si-NW FET for the sensing of proteins, which consisted of variable amounts of aspartic acid. The electrostatic potential profile, protein charge distributions, the response to various electrolyte concentration, and the impacts of various surface charge were studied by implementing the MPB model with the Si-NW FET biosensor. Additionally, a comparison between the use of the MPB and the Poisson–Boltzmann model in studying the effects of various surface charges was carried out. Taken together, it was found that the MPB model showed a higher resolution in studying the Si-NW FET biosensor model when higher concentrations and surface charges were administered.

Protein charge distribution in proteomes and its impact on translation.

As proteins are synthesized, the nascent polypeptide must pass through a negatively charged exit tunnel. During this stage, positively charged stretches can interact with the ribosome walls and slow the translation. Therefore, charged polypeptides may be important factors that affect protein expression. To determine the frequency and distribution of positively and negatively charged stretches in different proteomes, the net charge was calculated for every 30 consecutive amino acid residues, which corresponds to the length of the ribosome exit tunnel. The following annotated and reviewed proteins in the UniProt database (Swiss-Prot) were analyzed: 551,705 proteins from different organisms and a total of 180 million protein segments. We observed that there were more negative than positive stretches and that super-charged positive sequences (i.e., net charges ≥ 14) were underrepresented in the proteomes. Overall, the proteins were more positively charged at their N-termini and C-termini, and this feature was present in most organisms and subcellular localizations. To investigate whether the N-terminal charges affect the elongation rates, previously published ribosomal profiling data obtained from S. cerevisiae, without translation-interfering drugs, were analyzed. We observed a nonlinear effect of the charge on the ribosome occupancy in which values ≥ +5 and ≤ -6 showed increased and reduced ribosome densities, respectively. These groups also showed different distributions across 80S monosomes and polysomes. Basic polypeptides are more common within short proteins that are translated by monosomes, whereas negative stretches are more abundant in polysome-translated proteins. These findings suggest that the nascent peptide charge impacts translation and can be one of the factors that regulate translation efficiency and protein expression.