Model Protein System Research Articles

Effects of mono- and divalent- ions and their strength on foaming properties of infant formula protein model system

Metal ions may affect the foaming properties of infant formula products that including milk protein during reconstitution. In this study, an infant formula protein model system (IFPMS) composed of both whey protein and casein was constructed, and the effect of mono (Na+, K+) and divalent (Mg2+, Ca2+) ions and their strength (0–100 mM) on foaming properties was investigated. The mono ions had no significantly impact on foaming capacity (FC), MgCl2 slightly increase FC and reaching a maximum of 116.09 ± 7.46% at 25 mM, whereas CaCl2 significantly decreased FC from 105.78 ± 7.42% to 83.14 ± 6.75% as 0-25 mM (p < 0.05). Foam stability (FS) was significantly improved as all salts above 10 mM (p < 0.05). The dramatically decreased net charge and larger protein aggregates (> 25 mM) of IFPMS with divalent ions indicating their stronger charge-shielding effect, which caused slow adsorption of protein and increased surface tension. Multiple spectral results confirmed that salts enhanced protein interactions via inter-molecular and form a viscoelastic layer to stabilize foam. Absorbed protein and SDS-PAGE analysis revealed that MgCl2 promoted protein adsorption, while CaCl2 reduced casein in the foam phase. Correlation analysis further implied that the FC showed highly significant positive with absorbed protein, solubility, and β-turn (p ≤ 0.01), and FS was significantly correlated with secondary structure and viscosity (p ≤ 0.05). This study may provide useful information for an in-depth understanding of the foaming properties of infant formula.

Investigation of biomolecular dynamics by sensitivity-enhanced 1H–2H CPMAS NMR using matrix-free dynamic nuclear polarization

Molecular dynamics of functional groups contain valuable information about structural properties and functional activities in biomolecules. NMR spectroscopy is a sensitive tool for the investigation of molecular dynamics over a wide range of timescales and thus may deepen the understanding of the biomolecules of interest. Here, we present an approach to use DNP-enhanced 2H NMR to study dynamics of selectively deuterated methyl groups in insoluble proteins such as amyloid beta (Aβ) fibrils. We adopted and optimized the matrix-free DNP approach by varying the amount of added polarizing agent as well as the rehydration level of model proteins. We show that the DNP enhancement obtained in 1H–2H cross-polarization (CP) MAS spectra may increase the sensitivity for selectively deuterated Aβ fibril samples by more than one order of magnitude, accelerating the collection of spin-lattice relaxation data in the DNP-accessible temperature range between 100 and 150 K by up to 400-fold. However, below the coalescence temperature, which describes the transition from the fast to the slow exchange regime, the experimentally obtained relaxation time constants suffer from a paramagnetic relaxation enhancement effect due to the presence of the polarizing agent. This seems to be a general effect for biomolecules as it is also confirmed for two other protein model systems. Our demonstration opens the possibility to extend the scope of 2H NMR for dynamics measurements to effective concentrations and/or temperatures below what is currently accessible; however, the observed interplay between paramagnetic relaxation and molecular dynamics also emphasizes the necessity for a better understanding of relaxation effects in DNP-enhanced NMR.

Controlling heme redox properties in peptide amphiphile fibers with sequence and heme loading ratio

Controlling the reduction midpoint potential of heme B is a key factor in many bioelectrochemical reactions, including long-range electron transport. Currently, there are a number of globular model protein systems to study this biophysical parameter; however, there are none for large polymeric protein model systems (e.g., the OmcS protein from G. sulfurreducens). Peptide amphiphiles, short peptides with a lipid tail that polymerize into fibrous structures, fill this gap. Here, we show a peptide amphiphile model system where one can tune the electrochemical potential of heme B by changing the loading ratio and peptide sequence. Changing the loading ratio resulted in the most significant increase, with values as high as −22 mV down to −224 mV. Circular dichroism spectra of certain sequences show Cotton effects at lower loading ratios that disappear as more heme B is added, indicating an ordered environment that becomes disrupted if heme B is overpacked. These findings can contribute to the design of functional self-assembling biomaterials.

Constructing a strongly interacting Pea-Cod binary protein system by introducing metal cations toward enhanced gelling properties

Developing prospective plant-animal binary protein systems with desirable nutritional and rheological properties stands as a significant and challenging pursuit within the food industry. Our understanding of the effect of adding salt on the aggregation behavior of food proteins is currently based on single model protein systems, however, this knowledge is rather limited following binary protein systems. Herein, various ionic strength settings are used to mitigate the repulsive forces between pea-cod mixed proteins during the thermal process, which further benefits the construction of a strengthened gel network. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) collectively demonstrated that larger heat-induced protein aggregates were formed, which increased in size with higher ionic strength. In the presence of 2.5 mM CaCl2 and 50 mM NaCl, the disulfide bonds significantly increased from 19.3 to 27.53 and 30.5 μM/g, respectively. Notably, similar aggregation behavior could be found when introducing 2.5 mM CaCl2 or 25 mM NaCl, due to the enhanced aggregation tendency by specific binding of Ca2+ to proteins. With relevance to the strengthened cross-links between protein molecules, salt endowed composite gels with preferable gelling properties, evidenced by increased storage modulus. Additionally, the gelling temperature of mixed proteins decreased below 50 °C at elevated ionic strength. Simultaneously, the proportion of network proteins in composite gels increased remarkably from 82.05 % to 93.61 % and 92.31 % upon adding 5.0 mM CaCl2 and 100 mM NaCl, respectively. The findings provide a valuable foundation for designing economically viable and health-oriented plant-animal binary protein systems.

Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming β-propeller

Studies of folded-to-misfolded transitions using model protein systems reveal a range of unfolding needed for exposure of amyloid-prone regions for subsequent fibrillization. Here, we probe the relationship between unfolding and aggregation for glaucoma-associated myocilin. Mutations within the olfactomedin domain of myocilin (OLF) cause a gain-of-function, namely cytotoxic intracellular aggregation, which hastens disease progression. Aggregation by wild-type OLF (OLFWT) competes with its chemical unfolding, but only below the threshold where OLF loses tertiary structure. Representative moderate (OLFD380A) and severe (OLFI499F) disease variants aggregate differently, with rates comparable to OLFWT in initial stages of unfolding, and variants adopt distinct partially folded structures seen along the OLFWT urea-unfolding pathway. Whether initiated with mutation or chemical perturbation, unfolding propagates outward to the propeller surface. In sum, for this large protein prone to amyloid formation, the requirement for a conformational change to promote amyloid fibrillization leads to direct competition between unfolding and aggregation.

Effect of brewer's spent grain melanoidins on Maillard reaction products during storage of whey protein model systems

Maillard reaction readily takes place in dairy products because of the association between thermal treatments, extended storage and the matrix composition. Along with the impairment of protein digestion, the formation of glycation and α-dicarbonyl compounds is a concern for quality attributes of whey proteins when used as ingredients. In this paper, we outline the capacity of brewer's spent grain melanoidins in reducing the accumulation of α-dicarbonyl compounds, thus controlling the formation of dietary advanced glycation end-products in accelerated shelf life at 35 °C. Results revealed that brewer's spent grain melanoidins targeted methylglyoxal and glyoxal reactivity leading to the reduction of N-ε-carboxymethyllysine and methylglyoxal-hydroimidazolone up to 27 and 60%, respectively. We here describe that the presence of melanoidins is instrumental in limiting the undesired effects of α-dicarbonyl compounds on whey proteins.

Sulfated Polysaccharides as a Fighter with Protein Non-Physiological Aggregation: The Role of Polysaccharide Flexibility and Charge Density.

Proteins can lose native functionality due to non-physiological aggregation. In this work, we have shown the power of sulfated polysaccharides as a natural assistant to restore damaged protein structures. Protein aggregates enriched by cross-β structures are a characteristic of amyloid fibrils related to different health disorders. Our recent studies demonstrated that model fibrils of hen egg white lysozyme (HEWL) can be disaggregated and renatured by some negatively charged polysaccharides. In the current work, using the same model protein system and FTIR spectroscopy, we studied the role of conformation and charge distribution along the polysaccharide chain in the protein secondary structure conversion. The effects of three carrageenans (κ, ι, and λ) possessing from one to three sulfate groups per disaccharide unit were shown to be different. κ-Carrageenan was able to fully eliminate cross-β structures and complete the renaturation process. ι-Carrageenan only initiated the formation of native-like β-structures in HEWL, retaining most of the cross-β structures. In contrast, λ-carrageenan even increased the content of amyloid cross-β structures. Furthermore, κ-carrageenan in rigid helical conformation loses its capability to restore protein native structures, largely increasing the amount of amyloid cross-β structures. Our findings create a platform for the design of novel natural chaperons to counteract protein unfolding.

Effect of different salts on the foaming properties of model protein systems for infant formula

This multiscale study aimed to evaluate the effects of different salts (NaCl, KCl, MgCl2, and CaCl2) on the foaming capacity (FC) and foam stability (FS) of model protein systems (MPS) for infant formula via changes in surface and structural properties. Our results showed that the FC and FS of MPS were increased with the addition of NaCl, KCl, and MgCl2, whereas CaCl2 significantly decreased FC (79.5 ± 10.6%) and increased FS (93.2 ± 2.2%). The surface hydrophobicity was increased and the net charge and surface tension were reduced after the addition of salts. Structural analysis revealed the reduction of intensity of intrinsic fluorescence spectroscopy and UV absorption, and the conversion of α-helix into β-strand, which was attributed to protein agglomeration. Additionally, MgCl2 and CaCl2 exhibited larger size and lower net charge compared with NaCl and KCl, indicating a greater ability to bind to charged amino acids and form larger aggregates. Correlation analysis indicated that FC was positively related to adsorbed protein and β-turn and negatively correlated with particle size. In addition, FS showed a positive correlation with β-strand, apparent viscosity, and zeta potential. However, it exhibited a negative correlation with β-turn, α-helix, and sulfhydryl content. These results provide a theoretical reference for further understanding of the effect of salts on the foaming properties of MPS.

A probabilistic view of protein stability, conformational specificity, and design

Various approaches have used neural networks as probabilistic models for the design of protein sequences. These "inverse folding" models employ different objective functions, which come with trade-offs that have not been assessed in detail before. This study introduces probabilistic definitions of protein stability and conformational specificity and demonstrates the relationship between these chemical properties and the p(text {structure}|text {seq}) Boltzmann probability objective. This links the Boltzmann probability objective function to experimentally verifiable outcomes. We propose a novel sequence decoding algorithm, referred to as “BayesDesign”, that leverages Bayes’ Rule to maximize the p(text {structure}|text {seq}) objective instead of the p(text {seq}|text {structure}) objective common in inverse folding models. The efficacy of BayesDesign is evaluated in the context of two protein model systems, the NanoLuc enzyme and the WW structural motif. Both BayesDesign and the baseline ProteinMPNN algorithm increase the thermostability of NanoLuc and increase the conformational specificity of WW. The possible sources of error in the model are analyzed.

Antioxidant activities and emulsification properties of the new model systems of whey proteins and reduced sugars

The final products formed from the various systems of the Maillard reactions possess different functional properties such as browning intensity, antioxidant activity, and emulsion stability. To study these properties and activities, different systems of whey proteins reaction with glucose and fructose at different concentrations to form a new model system of Maillard reaction products (MRPs) was observed. Results showed that high optical densities (peaks) at 280 and 420 nm indicated the formation of the intermediate stages of MRPs and the formation of advanced MRPs, respectively. Additionally, results showed that these Maillard reaction model systems possessed different antioxidant activities as demonstrated by DPPH and reducing power assays (20 - 93.2% and 40 - 90%, respectively) depending on the type and concentration of sugar, and the incubation time. The whey protein-fructose model system possessed high antioxidant activity (93.2%), and had the highest percentage on the emulsion stability index (75.4%). The whey protein-fructose model systems comprised the highest number of the studied model systems to form MRPs, and had highly powerful antioxidant activity and emulsifying index.

Proximal Methionine Amino Acid Residue Affects the Properties of Redox-Active Tryptophan in an Artificial Model Protein.

Redox-active amino acid residues are at the heart of biological electron-transfer reactions. They play important roles in natural protein functions and are implicated in disease states (e.g., oxidative-stress-associated disorders). Tryptophan (Trp) is one such redox-active amino acid residue, and it has long been known to serve a functional role in proteins. Broadly speaking, there is still much to learn about the local features that make some Trp redox active and others inactive. Herein, we describe a new protein model system where we investigate how a methionine (Met) residue proximal to a redox-active Trp affects its reactivity and spectroscopy. We use an artificial variant of azurin from Pseudomonas aeruginosa to produce these models. We employ a series of UV-visible spectroscopy, electrochemistry, electron paramagnetic resonance, and density functional theory experiments to demonstrate the effect that placing Met near Trp radicals has in the context of redox proteins. The introduction of Met proximal to Trp lowers its reduction potential by ca. 30 mV and causes clear shifts in the optical spectra of the corresponding radicals. While the effect may be small, it is significant enough to be a way for natural systems to tune Trp reactivity.

Denaturation and Digestion Increase the Antioxidant Capacity of Proteins

It has been estimated and demonstrated that the antioxidant capacity of proteins is increased as a result of digestion in the gastrointestinal tract, which can be contributed by denaturation and digestion. This study aimed to evaluate the effect of denaturation and proteolytic digestion on the antioxidant activity of bovine serum albumin (BSA) and chicken egg white proteins in model systems. Denaturation with an anionic detergent (sodium dodecyl sulfate) and digestion with papain and trypsin increased the antioxidant activity/capacity of the proteins, apparently due to the increased exposure of amino acid residues responsible for the antioxidant activity of proteins (tyrosine, tryptophan, cysteine, histidine, arginine, and cystine in the ABTS● decolorization assay; cysteine, tryptophan, tyrosine, and cystine in the FRAP assay). As the increase in the protein antioxidant activity/capacity was limited in extent, it does not invalidate the use of the antioxidant capacity of proteins to be consumed as a rough measure of their antioxidant capacity after modifications in the gastrointestinal tract.

Structures of calmodulin–melittin complexes show multiple binding modes lacking classical anchoring interactions

Calmodulin (CaM) is a Ca2+ sensor protein found in all eukaryotic cells that regulates a large number of target proteins in a Ca2+ concentration-dependent manner. As a transient type hub protein, it recognizes linear motifs of its targets, though for the Ca2+-dependent binding no consensus sequence was identified. Its complex with melittin, a major component of bee venom, is often used as a model system of protein - protein complexes. Yet, the structural aspects of the binding are not well understood, as only diverse, low-resolution data are available concerning the association. We present the crystal structure of melittin in complex with Ca2+-saturated calmodulins from two, evolutionarily distant species, Homo sapiens and Plasmodium falciparum representing three binding modes of the peptide. Results - augmented by molecular dynamics simulations - indicate that multiple binding modes can exist for CaM-melittin complexes, as an intrinsic characteristic of the binding. While the helical structure of melittin remains, swapping of its salt bridges and partial unfolding of its C-terminal segment can occur. In contrast to the classical way of target recognition by CaM, we found that different sets of residues can anchor at the hydrophobic pockets of CaM, which were considered as main recognition sites. Finally, the nanomolar binding affinity of the CaM-melittin complex is created by an ensemble of arrangements of similar stability - tight binding is achieved not by optimized specific interactions but by simultaneously satisfying less optimal interaction patterns in co-existing different conformers.

Formation of Nε-Carboxymethyl-Lysine and Nε-Carboxyethyl-Lysine in Heated Fish Myofibrillar Proteins with Glucose: Relationship with Its Protein Structural Characterization.

The formation of advanced glycation end products (AGEs), including Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL), in a fish myofibrillar protein and glucose (MPG) model system at 80 °C and 98 °C for up to 45 min of heating were investigated. The characterization of protein structures, including their particle size, ζ-potential, total sulfhydryl (T-SH), surface hydrophobicity (H0), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR), were also analyzed. It was found that the covalent binding of glucose and myofibrillar protein at 98 °C promoted protein aggregation when compared with the fish myofibrillar protein (MP) heated alone, and this aggregation was associated with the formation of disulfide bonds between myofibrillar proteins. Furthermore, the rapid increase of CEL level with the initial heating at 98 °C was related to the unfolding of fish myofibrillar protein caused by thermal treatment. Finally, correlation analysis indicated that the formation of CEL and CML had a significantly negative correlation with T-SH content (r = -0.68 and r = -0.86, p ≤ 0.011) and particle size (r = -0.87 and r = -0.67, p ≤ 0.012), but was weakly correlated with α-Helix, β-Sheet and H0 (r2 ≤ 0.28, p > 0.05) during thermal treatment. Overall, these findings provide new insights into the formation of AGEs in fish products based on changes of protein structure.

Binding of E. coli single-stranded DNA binding protein to damaged DNA substrates measured at single molecule level using AFM.

Single-stranded DNA binding proteins (SSBs) are essential cellular components, binding to transiently exposed regions of single-stranded DNA (ssDNA) with high affinity and sequence non-specificity to coordinate DNA repair and replication. E. coli SSB (Ec SSB), a homotetramer that wraps variable lengths of ssDNA in multiple conformations, is a model protein system with resolved structures that is well-studied across experimental conditions (temperature, salt, pH, substrate length, etc.). We use atomic force microscopy (AFM) to investigate the binding of SSB to individual ssDNA molecules. Furthermore, we introduce noncanonical DNA units that mimic naturally occurring DNA damage (synthetic abasic sites and non-DNA linker) into our experimental constructs at sites predicted to interact with binding Ec SSB. By measuring the fraction of DNA constructs with SSB bound as well as the number of SSB proteins bound per construct, we determine the protein binding affinity and cooperativity. We find that both the affinity and cooperativity of protein binding are affected by DNA damage. Changes in the binding and cooperative behaviors of SSB proteins across these constructs can inform how genomic repair and replication processes may change as environmental damage accumulates.

Capturing the influence of a crowded environment on protein GB1 dimerization at coarse-grained resolutions.

In-cell protein-protein association, which is crucial in enzyme catalysis and polymerization, occurs in an environment that is highly heterogeneous and crowded. The crowder molecules exclude the reactant molecules from occupying certain regions of the cell, resulting in changes in the reaction thermodynamics and kinetics. Recent studies, both experiment and simulations, also revealed that the nature of interaction between crowder and protein species, in particular the soft interactions, plays an important role in crowder induced effects on protein association. To this end, from a simulation perspective, it is important to decipher the level of structural resolution in a protein-crowder model that can faithfully capture the influences of crowding on protein association. Here we investigate a model protein system in a dilute medium as well as in a crowded medium, adopting two structural resolutions of the constituent molecules, and using either molecular or Langevin dynamics to propagate the equations of motion. In particular, we look at dimerization of protein GB1, since GB1 dimers can exist in both side-by-side and domain-swapped conformations, showing the important role of residue-level structural details. The free energy change in protein association in a crowded solution relative to a crowder free medium obtained from the simulations is then compared with experimental results. The results reveal the pros and cons of adopting different resolution approaches.

Impact of Hydrophobic Chains in Five-Coordinate Glucoconjugate Pt(II) Anticancer Agents.

This study describes new platinum(II) cationic five-coordinate complexes (1-R,R') of the formula [PtR(NHC)(dmphen)(ethene)]CF3SO3 (dmphen = 2,9-dimethyl-1,10-phenanthroline), containing in their axial positions an alkyl group R (methyl or octyl) and an imidazole-based NHC-carbene ligand with a substituent R' of variable length (methyl or octyl) on one nitrogen atom. The Pt-carbene bond is stable both in DMSO and in aqueous solvents. In DMSO, a gradual substitution of dmphen and ethene is observed, with the formation of a square planar solvated species. Octanol/water partitioning studies have revealed the order of hydrophobicity of the complexes (1-Oct,Me > 1-Oct,Oct > 1-Me,Oct > 1-Me,Me). Their biological activity was investigated against two pairs of cancer and non-cancer cell lines. The tested drugs were internalized in cancer cells and able to activate the apoptotic pathway. The reactivity of 1-Me,Me with DNA and protein model systems was also studied using UV-vis absorption spectroscopy, fluorescence, and X-ray crystallography. The compound binds DNA and interacts in various ways with the model protein lysozyme. Remarkably, structural data revealed that the complex can bind lysozyme via non-covalent interactions, retaining its five-coordinate geometry.

Theory and Applications of Nitroxide-based Paramagnetic Cosolutes for Probing Intermolecular and Electrostatic Interactions on Protein Surfaces.

Solvent paramagnetic relaxation enhancement (sPRE) arising from nitroxide-based cosolutes has recently been used to provide an atomic view of cosolute-induced protein denaturation and to characterize residue-specific effective near-surface electrostatic potentials (ϕENS). Here, we explore distinct properties of the sPRE arising from nitroxide-based cosolutes and provide new insights into the interpretation of the sPRE and sPRE-derived ϕENS. We show that: (a) the longitudinal sPRE rate Γ1 is heavily dependent on spectrometer field and viscosity, while the transverse sPRE rate Γ2 is much less so; (b) the spectral density J(0) is proportional to the inverse of the relative translational diffusion constant and is related to the quantity ⟨r-4⟩norm, a concentration-normalized equilibrium average of the electron-proton interspin separation; and (c) attractive intermolecular interactions result in a shortening of the residue-specific effective correlation time for the electron-proton vector. We discuss four different approaches for evaluating ϕENS based on Γ2, J(0), Γ1, or ⟨r-6⟩norm. The latter is evaluated from the magnetic field dependence of Γ1 in conjunction with Γ2. Long-range interactions dominate J(0) and Γ2, while, at high magnetic fields, the contribution of short-range interactions becomes significant for J(ω) and hence Γ1; the four ϕENS quantities enable one to probe both long- and short-range electrostatic interactions. The experimental ϕENS potentials were evaluated using three model protein systems, two folded (ubiquitin and native drkN SH3) and one intrinsically disordered (unfolded state of drkN SH3), in relation to theoretical ϕENS potentials calculated from atomic coordinates using the Poisson-Boltzmann theory with either a r-6 or r-4 dependence.

Antibacterial Activities, Chemical Composition, and Efficacy of Green Extract Carica Papaya Peel on Food Model Systems

This study investigated anti-bacterial activities, chemical composition, and extract efficacy of Carica papaya peel (CPPE) var. Sekaki/Hong Kong. Nine green solvents were used to extract the Carica papaya peel, and the extracts were subjected to anti-bacterial tests and assays against 14 bacteria. The most potent extract was then subjected to phenolic and flavonoid assays, gas chromatography-mass spectrometry (GC/MS) analysis, and efficacy study on food model systems. All CPPEs showed anti-bacterial activities, and pentane extract had moderate to high anti-bacterial activities against all 14 bacteria. Ethanol extract of Carica papaya peel (ECPPE) inhibited C. perfringens, L. monocytogenes, B. subtilis, V. parahaemolyticus, and V. vulnificus with a minimum inhibitory concentration (MIC) of 1.563 mg/ml; therefore, the ECPPE was selected as the most potent extract. The total phenolic (TPC) and flavonoid contents (TFC) of the CPPEs ranged between 6.20 to 58.75 mg GAE/g DW and 1.35 - 29.09 mg QE/g DW, respectively. Palmitic acid, linoleic acid, β-sitosterol, and stigmasterol in ECPPE may be potential anti-bacterial compounds that render anti-bacterial activities. This study evaluated the ECPPE effectiveness on carbohydrate, protein, fat, and fibre model systems via optical density measurement against C. perfringens, L. monocytogenes, B. subtilis, V. parahaemolyticus, and V. vulnificus. The result showed that the ECPPE could effectively inhibit the tested bacteria in low carbohydrate and high protein, fat, and fibre food model systems.

Sequence grammar underlying the unfolding and phase separation of globular proteins

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.