Protein Surface Charge Research Articles

Tripeptide Self-Assembled Monolayers as Biocompatible Surfaces for Cytochrome c Electrochemistry.

Biocompatible tripeptide self-assembled monolayers (SAMs) are designed with a carboxylate group on the terminal amino acid (glutamate, aspartate, or amino adipate) to electrostatically attract the lysine groups around the heme crevice in horse heart cytochrome c (cyt c), creating an electroactive protein/tripeptide/Au interfacial structure. Exposing the peptide/Au electrode to cyt c resulted in an 11 ± 3 pmol/cm2 electroactive protein surface coverage. Topographical images of the interfacial structure are obtained down to single-protein resolution by atomic force microscopy. Uniform protein monolayer assemblies are formed on the Au electrode with no major surface roughness changes. The cyt c/peptide/Au electrode systems were examined electrochemically to probe surface charge effects on the redox thermodynamics and kinetics of cyt c. Neutralization of protein surface charge due to adsorption on anionic COOH-terminated SAMs was found to change the formal potential, as determined by cyclic voltammetry. The cyt c/peptide/Au electrodes exhibit formal potentials shifted to more positive values, have a surface carboxylic acid pKa of 6 or higher, and produce effective cyt c surface charges (Zox) of -6 to -14. The Marcus theory is utilized to determine the protein electron transfer rates, which are ∼5 times faster for cyt c/tripeptide/Au compared to cyt c/11-mercaptoundecanoic acid SAMs of similar chain lengths.

The effects of germination on the composition and functional properties of hemp seed protein isolate

The effects of germination on the functional properties of hemp seed protein isolates was evaluated in this study. Significant seed storage protein degradation was observed after three days of germination, while protein surface charge was altered after one day of germination. Protein solubility increased in neutral to slightly acidic pH, with 5-day germination increasing hemp seed protein solubility from 10.3% to 21.8% at pH 7.0. Water holding capacity increased from 1.03 g H 2 O/g isolate to 1.34 g H 2 O/g isolate while oil holding capacity stayed relatively constant. Germinated protein isolates demonstrated higher foaming capacity (69.2%–111.2%) and foaming stability (53.6%–75.1%) than non-germinated protein isolates. Five-day germinated hemp seed protein isolate was the only adequate emulsifier in this study, capable of stabilizing emulsion droplets down to 2.90 μm in diameter. Hemp seed protein formed self-supporting gels at minimum of 2 or 3 w/v %, and germination increased the hardness of hemp seed protein gels (38.02 g–49.12 g for 10 w/v % gels). These findings indicate the ability to leverage germination as a clean-label process for improving the functional properties of hemp seed protein. • Hemp seed storage proteins are degraded after three days of germination. • Germinated hemp seed protein isolates are more soluble near a neutral pH. • Germination improves hemp protein WHC, foaming, emulsifying, and gelling properties. • LGC for hemp protein at pH 7 was determined at 2–3 w/v %.

Functional evolution of SARS-CoV-2 spike protein: Maintaining wide host spectrum and enhancing infectivity via surface charge of spike protein

The SARS-CoV-2 virus, which causes the COVID-19, is rapidly accumulating mutations to adapt to the hosts. We collected SARS-CoV-2 sequence data from the end of 2019 to January 2023 to analyze for their evolutionary features during the pandemic. We found that most of the SARS-CoV-2 genes are undergoing negative purifying selection, while the spike protein gene (S-gene) is undergoing rapid positive selection. From the original strain to the alpha, delta and omicron variant types, the Ka/Ks of the S-gene increases, while the Ka/Ks within one variant type decreases over time. During the evolution, the codon usage did not evolve towards optimal translation and protein expression. In contrast, only S-gene mutations showed a remarkable trend on accumulating more positive charges. This facilitates the infection via binding human ACE2 for cell entry and binding furin for cleavage. Such a functional evolution emphasizes the survival strategy of SARS-CoV-2, and indicated new druggable target to contain the viral infection. The nearly fully positively-charged interaction surfaces indicated that the infectivity of SARS-CoV-2 virus may approach a limit.

Intracellular phase separation of globular proteins facilitated by short cationic peptides

Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We propose adding to this toolbox designer peptide tags that provide several potential advantages for engineering protein phase separation in bacteria. Herein, we demonstrate the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates and provide a foundational study for their development as tools for condensate engineering. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation in E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active.

Lattice-model analysis of the effect of protein surface charge distribution on amorphous aggregation and condensation

We describe a lattice model to analyze the effect of protein surface charge distributions on protein solubility, aggregation, and condensation. Surface charges were modeled by considering a protein as a cubic particle with each face covered by two negative and positive patches (net zero charge). Particles were moved to adjacent lattice points according to a Monte Carlo algorithm. We observed that proteins having the same isoelectric point can have distinct aggregation propensities, depending on surface charge arrangements. Furthermore, with regard to the effect of heterogeneous intracellular environment, mixing two particle types affected the aggregation properties of the individual type.

Influence of protein configuration on aggregation kinetics of nanoplastics in aquatic environment

Aggregation kinetics of nanoplastics in aquatic environment are influenced by their interactions with proteins having different structures and properties. This study employed time-resolved dynamic light scattering (TR-DLS) to investigate the effects of 5 proteins (bovine hemoglobin (BHb), bovine (BSA) and human serum albumin (HSA), collagen type I (Col I), and bovine casein (CS)) on aggregation kinetics of polystyrene nanoplastics (PSNPs) under natural water conditions, which were simulated using various ionic strength (1-1000 mM NaCl and 0.01-100 mM CaCl2), pH (3-9), and protein concentration (1-5 mg/L of total organic carbon). The results indicated that the interactions between proteins and PSNPs strongly depended on electrostatic properties, protein structures, and solution chemistries, which induced distinct aggregation behaviors in NaCl and CaCl2 solutions. Electrostatic repulsion and steric hindrance dominated their interactions in NaCl solution by stabilizing PSNPs with the order of spherical BSA and disordered CS > heart-shaped HSA > fibrillar Col I; whereas positively charged BHb destabilized PSNPs with aggregation rate of 1.71 nm/s at 300 mM NaCl. In contrast, at CaCl2 concentration below 20 mM, proteins destabilized PSNPs following the sequence of HSA > BHb > Col I > BSA depending on counterbalance among double layer compression, cation bridging, and steric hindrance; whereas CS stabilized PSNPs by precipitating Ca2+ that inhibited charge screening effect. Both protein concentration and solution pH affected protein corona formation, surface charge, and protein structure that altered stability of PSNPs. Characterizations using fluorescence spectroscopy, circular dichroism, and two-dimensional correlation analysis spectroscopy showed fluorescence quenching and ellipticity reduction of proteins, indicating strong adsorption affinity between PSNPs and proteins. The study provides insight to how protein configuration and water chemistry affect fate and transport of nanoplastics in aquatic environment.

Tuning Strain Stiffening of Protein Hydrogels by Charge Modification.

Strain-stiffening properties derived from biological tissue have been widely observed in biological hydrogels and are essential in mimicking natural tissues. Although strain-stiffening has been studied in various protein-based hydrogels, effective approaches for tuning the strain-stiffening properties of protein hydrogels have rarely been explored. Here, we demonstrated a new method to tune the strain-stiffening amplitudes of protein hydrogels. By adjusting the surface charge of proteins inside the hydrogel using negatively/positively charged molecules, the strain-stiffening amplitudes could be quantitively regulated. The strain-stiffening of the protein hydrogels could even be enhanced 5-fold under high deformations, while the bulk property, recovery ability and biocompatibility remained almost unchanged. The tuning of strain-stiffening amplitudes using different molecules or in different protein hydrogels was further proved to be feasible. We anticipate that surface charge adjustment of proteins in hydrogels represents a general principle to tune the strain-stiffening property and can find wide applications in regulating the mechanical behaviors of protein-based hydrogels.

The potential of white and soft-rot fungi for biodegradation of multi-walled carbon nanotubes (MWCNTs): characterization and enzyme analysis

ABSTRACT Multi-walled carbon nanotubes (MWCNTs) have significant environmental concerns for soil, water and ecosystems. Since their harmful effects on human health, including respiratory disease, they are considered dangerous substances. Thus, it is required to discover a constructive solution to decrease of MWCNT toxicity. Due to their high degradability, white rot and soft rot fungi have been used in the biodegradation of various pollutants. In this study, we evaluated the ability of native Iranian white and soft rot fungi to degrade MWCNTs mainly by means of dynamic light scattering (DLS) and surface charge measurement. In this regard, growth kinetics, CO2 and protein production, size, surface charge and pH change were measured. Continuous Raman spectroscopy, scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging were carried out to detect the process of degradation. The results showed that the morphology, arrangement, size and diameter of MWCNTs were modified due to fungal degradation. The cytotoxicity of treated MWCNTs was determined by the MTT test. The results indicated that native Iranian white and soft rot fungi have a significant ability for degradation of MWCNTs. Furthermore, it could be concluded that fungal treatment could reduce the toxicity of MWCNTs and the best result was achieved in 500 ppm MWCNTs for Trichoderma sp. The activities of oxidative enzymes such as laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) were measured to determine the mechanism of degradation. Enzyme assays have suggested that these oxidising enzymes especially laccase might play a key role in the degradation of MWCNTs in white rot and soft rot fungi. The overall results confirmed the reproductive ability of Trichoderma sp. WF29 as a soft rot and two white rot fungi Irpex lacteus WF36 and Trametes versicolor to degrade MWCNT.

Detecting Individual Proteins and Their Surface Charge Variations in Solution by the Potentiometric Nanoimpact Method.

Label-free detection and analysis of proteins in their natural form and their dynamic interactions with substrates at the single-molecule level are important for both fundamental studies and various applications. Herein, we demonstrate a simple potentiometric method to achieve this goal by detecting the native charge of protein in solution by utilizing the principle of single-entity electrochemistry techniques. When a charged protein moves near the vicinity of a floating carbon nanoelectrode connected to a high-impedance voltage meter, the distinct local electrostatic potential changes induced by the transient collision event of protein, also called the "nanoimpact" event, can be captured by the nanoelectrode as a potential probe. This potentiometric method is highly sensitive for charged proteins, and low-molecular-weight proteins less than 10 kDa can be detected in low-salt-concentration electrolytes. By analyzing the shape and magnitude of the recorded time-resolved potential change and its time derivative, we can reveal the charge and motion of the protein in the nonspecific protein-surface interaction event. The charge polarity variations of the proteins at different pH values were also successfully probed. Compared with synthetic spherical nanoparticles, the statistical analysis of many single-molecule nanoimpact events revealed a large variation in the recorded transient potential signals, which may be attributed to the intrinsic protein dynamics and surface charge heterogeneity, as suggested by the finite element method and molecular dynamic simulations.

Improvement of the halotolerance of a Bacillus serine protease by protein surface engineering.

A moderately halotolerant serine protease was previously isolated from Bacillus subtilis from salted, fermented food. Eight mutation sites on the protein surface were selected for protein engineering based on sequence and structural comparisons with moderately halotolerant proteases and homologous non-halotolerant proteases. The newly constructed multiple mutants with substituted Asp and Arg residues were compared with the recombinant wild type (rApr) and the previously constructed mAla-8 substituted with Ala to analyze the contribution of protein surface charge to the salt adaptation of the protease. The three mutants showed >1.2-fold greater halotolerance than rApr. In addition, the mutants showed a broader range of pH stability than rApr, retaining >80% of their maximum activity in the pH range 5.0-11. The mutants also retained >75% of their activity after incubation for 1 h at pH 8.0 and 55°C or at pH 11.5 and 25°C. The Asp and Arg residues exchanged by multiple substitution probably played a role in increasing protein surface hydration and solubility in high salt conditions. This study illustrated that increasing a high proportion of the negative or positive charge on the surface of the Bacillus serine protease stably improved the protein's salt adaptation.

Distinct phosphoinositides define the biotrophic interface of plant–microbe interactions

Distinct phosphoinositides define the biotrophic interface of plant–microbe interactions

Direct comparison of the tofu-like precipitate formation by adding different coagulants: magnesium chloride and glucono-δ-lactone

Tofu is produced by adding a coagulant such as MgCl2 and glucono-δ-lactone (GDL) in soymilk. However, the molecular mechanism of tofu formation by adding these coagulants has been compared between the results obtained under different conditions. In this study, the formation of a tofu-like precipitate (TLP) by adding GDL was directly compared with that formed by adding MgCl2 under the same conditions except for the coagulants. The effects of both the coagulants were almost the same on the changes in the precipitate weight, supernatant protein concentration, and urea-soluble protein concentration, indicating that a common contributing factor induces TLP formation. However, the effects of the coagulants on pH were largely different, suggesting that pH reduction is not an absolute requirement in TLP formation induced by adding MgCl2. Moreover, the findings of this study revealed that the decrease in the surface charge of soymilk proteins is a common initiation factor for TLP formation, whereas the intermolecular hydrophobic interaction is an important factor for the formation of urea-insoluble precipitates. Overall, these findings will be useful in discovering new coagulants to enhance the quality characteristics of tofu.

Examining the amino acid composition, secondary structure, and physicochemical and functional properties of proteins isolated from local lentil landraces of Anatolia

AbstractBackground and objectivesProviding information on protein structure and functionality is essential for evaluating the potential of specific agricultural commodities to be utilized as plant‐based protein sources. Amino acid composition, secondary structure, surface charge, thermal, and functional properties of proteins isolated from five local lentil landraces were investigated. Secondary objective of the study was to investigate if a relationship exists between the amino acid composition and the physicochemical or functional properties of the isolates.FindingsAspartic acid and glutamic acid were found to be the dominant amino acids in the isolates while they were poor in methionine. Denaturation temperatures of isolates ranged between 96–108°C and their secondary structure mainly consisted of β‐sheet. Emulsion capacity, emulsifying activity (EAI), and emulsion stability indices (ESI) of isolates were found to be between 403.8–468.2 g oil/g protein, 18.8–24.2 m2/g, and 43.4–125.5 min, respectively. Foaming capacity of the isolates changed between 71% and 91% whereas their least gelation concentration varied between 110 and 150 g/L.ConclusionsYusufhan isolate which had relatively higher acidic amino acid content showed the highest net charge and solubility at acidic pH. Tigris isolate which had higher hydrophobic amino acid content compared to the other isolates showed relatively high thermal stability, EAI and ESI.Significance and noveltyFindings of the study make a contribution toward the potential use of local lentil landraces as sources for extracting protein‐based ingredients.

Probing the Mechanism of Antibody-Triggered Aggregation of Gold Nanoparticles.

The unique physicochemical properties of gold nanoparticles (AuNPs) provide many opportunities to develop novel biomedical technologies. The surface chemistry of AuNPs can be engineered to perform a variety of functions, including targeted binding, cellular uptake, or stealthlike properties through the immobilization of biomolecules, such as proteins. It is well established that proteins can spontaneously adsorb onto AuNPs, to form a stable and functional bioconjugate; however, the protein-AuNP interaction may result in the formation of less desirable protein-AuNP aggregates. Therefore, it is imperative to investigate the protein-AuNP interaction and elucidate the mechanism by which protein triggers AuNP aggregation. Herein, we systematically investigated the interaction of immunoglobulin G (IgG) antibody with citrate-capped AuNPs as a function of solution pH. We found that the addition of antibody triggers the aggregation of AuNPs for pH < 7.5, whereas a monolayer of antibody adsorbs onto the AuNP to form a stable bioconjugate when the antibody is added to AuNPs at pH ≥ 7.5. Our data identifies electrostatic bridging between the antibody and the negatively charged AuNPs as the mechanism by which aggregation occurs and rules out protein unfolding and surface charge depletion as potential causes. Furthermore, we found that the electrostatic bridging of AuNPs is reversible within the first few hours of interaction, but the protein-AuNP interactions strengthen over 24 h, after which the protein-AuNP aggregate is irreversibly formed. From this data, we developed a straightforward approach to acrylate the basic residues on the antibody to prevent protein-induced aggregation of AuNP over a wide pH range. The results of this study provide additional insight into antibody-nanoparticle interactions and provide a pathway to control the interaction with the potential to enhance the conjugate function.

The Dawn of Metal-Oxo Clusters as Artificial Proteases: From Discovery to the Present and Beyond.

The selective cleavage of peptide bonds in proteins is of paramount importance in many areas of the biological and medical sciences, playing a key role in protein structure/function/folding analysis, protein engineering, and targeted proteolytic drug design. Current applications that depend on selective protein hydrolysis largely rely on costly proteases such as trypsin, which are sensitive to the pH, ionic strength, and temperature conditions. Moreover, >95% of peptides deposited in databases are generated from trypsin digests, restricting the information within the analyzed proteomes. On the other hand, harsh and toxic chemical reagents such as BrCN are very active but cause permanent modifications of certain amino acid residues. Consequently, transition-metal complexes have emerged as smooth and selective artificial proteases owing to their ability to provide larger fragments and complementary structural information. In the past decade, our group has discovered the unique protease activity of diverse metal-oxo clusters (MOC) and pioneered a distinctive approach to the development of selective artificial proteases. In contrast to classical coordination complexes which often depend on amino acid side chains to control the regioselectivity, the selectivity profile of MOCs is determined by a complex combination of structural factors, such as the protein surface charge, metal coordination to specific side chains, and hydrogen bonding between the protein surface and the MOC scaffold.In this Account, we present a critical overview of our detailed kinetic, spectroscopic, and crystallographic studies in MOC-assisted peptide bond hydrolysis, from its origins to the current rational and detailed mechanistic understanding. To this end, reactivity trends related to the structure and properties of MOCs based on the hydrolysis of small model peptides and key structural aspects governing the selectivity of protein hydrolysis are presented. Finally, our endeavors in seeking the next generation of heterogeneous MOC-based proteases are briefly discussed by embedding MOCs in metal-organic frameworks or using them as discrete nanoclusters in the development of artificial protease-like materials (i.e., nanozymes). The deep and comprehensive understanding sought experimentally and theoretically over the years in aqueous systems with intrinsic polar and charged substrates provides a unique view of the reactivity between inorganic moieties and biomolecules, thereby broadly impacting several different fields (e.g., catalysis in biochemistry, inorganic chemistry, and organic chemistry).

Hydrogen-Bond Dynamics and Energetics of Biological Water.

Water molecules in the immediate vicinity of biomacromolecules and biomimetic organized assemblies often exhibit a markedly distinct behavior from that of their bulk counterparts. The overall sluggish behavior of biological water substantially affects the stability and integrity of biomolecules, as well as the successful execution of various crucial water-mediated biochemical phenomena. In this Minireview, insights are provided into the features of truncated hydrogen-bond networks that grant biological water its unique characteristics. In particular, experimental results and theoretical investigations, based on chemical kinetics, are presented that have shed light on the dynamics and energetics governing such characteristics. It is emphasized how such details help us to understand the energetics of biological water, an aspect relatively less explored than its dynamics. For instance, when biological water at hydrophilic or charged protein surfaces was explored, the free energy of H-bond breakage was found to be of the order of 0.4 kcal mol-1 higher than that of bulk water.

Protein Vesicles Self-Assembled from Functional Globular Proteins with Different Charge and Size.

Protein vesicles can be synthesized by mixing two fusion proteins: an elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) with a globular, soluble protein fused to a glutamate-rich leucine zipper (ZE). Currently, only fluorescent proteins have been incorporated into vesicles; however, for protein vesicles to be useful for biocatalysis, drug delivery, or biosensing, vesicles must assemble from functional proteins that span an array of properties and functionalities. In this work, the globular protein was systematically changed to determine the effects of the surface charge and size on the self-assembly of protein vesicles. The formation of microphases, which included vesicles, coacervates, and hybrid structures, was monitored at different assembly conditions to determine the phase space for each globular protein. The results show that the protein surface charge has a small effect on vesicle self-assembly. However, increasing the size of the globular protein decreases the vesicle size and increases the stability at lower ZE/ZR molar ratios. The phase diagrams created can be used as guidelines to incorporate new functional proteins into vesicles. Furthermore, this work reports catalytically active enzyme vesicles, demonstrating the potential for the application of vesicles as biocatalysts or biosensors.

Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation.

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe3 O4 . A thorough investigation of protein-MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe3 O4 , it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein-MNP interaction but also sheds light on the molecular mechanism underling this process.

Role of Physicochemical Properties of Protein in Modulating the Nanoparticle-Bio Interface.

Nanoparticles, on exposure to the biological milieu, tend to interact with macromolecules to form a biomolecular corona. The biomolecular corona confers a unique biological identity to nanoparticles, and its protein composition plays a deterministic role in the biological fate of nanoparticles. The physiological behavior of proteins stems from their physicochemical properties, including surface charge, hydrophobicity, and structural stability. However, there is insufficient understanding about the role of physicochemical properties of proteins in biomolecular corona formation. We hypothesized that the physicochemical properties of proteins would influence their interaction with nanoparticles and have a deterministic effect on nanoparticle-cell interactions. To test our hypothesis, we used model proteins from different structural classes to understand the effect of secondary structure elements of proteins on the nanoparticle-protein interface. Further, we modified the surface of proteins to study the role of protein surface characteristics in governing the nanoparticle-protein interface. For this study, we used mesoporous silica nanoparticles as a model nanoparticle system. We observed that the surface charge of proteins governs the nature of the primary interaction and the extent of subsequent secondary interactions causing structural rearrangements of the protein. We also observed that the secondary structural contents of proteins significantly affected both the extent of secondary interactions at the nanoparticle-protein interface and the dispersion state of the nanoparticle-protein complex. Further, we studied the interactions of different protein-coated nanoparticles with different cells (fibroblast, carcinoma, and macrophage). We observed that different cells internalized the nanoparticle-protein complex as a function of secondary structural components of the protein. The type of model protein had a significant effect on their internalization by macrophages. Overall, we observed that the physicochemical characteristics of proteins had a significant role in modulating the nanoparticle-bio-interface at the level of both biomolecular corona formation and nanoparticle internalization by cells.

Specific Buffer Effects on the Intermolecular Interactions among Protein Molecules at Physiological pH.

BSA and lysozyme molecular motion at pH 7.15 is buffer-specific. Adsorption of buffer ions on protein surfaces modulates the protein surface charge and thus protein-protein interactions. Interactions were estimated by means of the interaction parameter kD obtained from plots of diffusion coefficients at different protein concentrations (Dapp = D0[1 + kDCprotein]) via dynamic light scattering and nuclear magnetic resonance. The obtained results agree with recent findings confirming doubts regarding the validity of the Henderson-Hasselbalch equation, which has traditionally provided a basis for understanding pH buffers of primary importance in solution chemistry, electrochemistry, and biochemistry.