Improving common vetch protein isolate flavor through glutaminase-mediated deamidation.

Common vetch protein, similar to pea protein, offers valuable qualities like being non-GMO, hypoallergenic, and nutritious. However, its strong beany flavor hinders consumer acceptance. This study explores enzymatic deamidation using glutaminase to address this issue. GC-MS analysis identified 54 volatile compounds in the raw material protein, with 2-pentylfuran, hexanal, and several nonenals contributing the most to the undesirable aroma. Principal component analysis (PCA) confirmed the effectiveness of glutaminase deamidation in removing these off-flavors. The study further reveals that deamidation alters the protein's secondary structure, with an increase in α - helix structure and a decrease in ß - sheet structure. The surface hydrophobicity increased from 587.33 ± 2.63 to 1855.63 ± 3.91 exposing hydrophobic clusters that bind flavor compounds. This disruption weakens the interactions that trap these undesirable flavors, ultimately leading to their release and a more pleasant aroma. These findings provide valuable insights for enzymatic deodorization of not only common vetch protein but also pea protein.

Dominant Chemical Interactions Governing the Folding Mechanism of Oligopeptides.

The hydrophobic effect is the main factor that drives the folding of polypeptide chains. In this study, we have examined the influence of the hydrophobic effect in the context of the main mechanical forces approach, mainly in relation to the establishment of specific interplays, such as hydrophobic and CH-π cloud interactions. By adopting three oligopeptides as model systems to assess folding features, we demonstrate herein that these finely tuned interactions dominate over electrostatic interactions, including H-bonds and electrostatic attractions/repulsions. The folding mechanism analysed here demonstrates cooperation at the single-residue level, for which we propose the terminology of "single residues cooperative folding". Overall, hydrophobic and CH-π cloud interactions produce the main output of the hydrophobic effect and govern the folding mechanism, as demonstrated in this study with small polypeptide chains, which in turn represent the main secondary structures in proteins.

Nanoscale Structural Characterization of Amyloid ß 1-42 Oligomers and Fibrils Grown in the Presence of Fatty Acids.

Mono- and polyunsaturated fatty acids (FAs) are broadly used as food supplements. However, their effect on the aggregation of amyloidogenic proteins remains unclear. In this study, we investigated the effect of a large number of mono- and polyunsaturated, as well as fully saturated FAs on the aggregation of amyloid ß1-42 (Aß1-42) peptide. A progressive aggregation of this peptide is the expected molecular cause of Alzheimer's disease (AD), one of the most common neurodegenerative pathologies in the world. We found that arachidonic and stearic acids delayed the aggregation of Aß1-42. Using Nano-Infrared spectroscopy, we found that FAs caused very little if any changes in the secondary structure of Aß1-42 oligomers and fibrils formed at different stages of protein aggregation. However, the analyzed mono- and polyunsaturated, as well as fully saturated FAs uniquely altered the toxicity of Aß1-42 fibrils. We found a direct relationship between the degree of FAs unsaturation and toxicity of Aß1-42 fibrils formed in their presence. Specifically, with an increase in the degree of unsaturation, the toxicity Aß1-42/FA fibrils increased. These results indicate that fully saturated or monounsaturated FAs could be used to decrease the toxicity of amyloid aggregates and, consequently, decelerate the development of AD.

Secondary structure propensities of the Ebola delta peptide E40 in solution and model membrane environments.

The Ebola delta peptide is an amphipathic, 40-residue peptide encoded by the Ebola virus, referred to as E40. The membrane-permeabilising activity of the E40 delta peptide has been demonstrated in cells and lipid vesicles suggesting the E40 delta peptide likely acts as a viroporin. The lytic activity of the peptide increases in the presence of anionic lipids and a disulphide bond in the C-terminal part of the peptide. Previous in silico work predicts the peptide to show a partially helical structure, but there is no experimental information on the structure of E40. Here, we use circular dichroism spectroscopy to report the secondary structure propensities of the reduced and oxidised forms of the E40 peptide in water, detergent micelles, and lipid vesicles composed of neutral and anionic lipids (POPC and POPG, respectively). Results indicate that the peptide is predominately a random coil in solution, and the disulphide bond has a small but measurable effect on peptide conformation. Secondary structure analysis shows large uncertainties and dependence on the reference data set and, in our system, cannot be used to accurately determine the secondary structure motifs of the peptide in membrane environments. Nevertheless, the spectra can be used to assess the relative changes in secondary structure propensities of the peptide depending on the solvent environment and disulphide bond. In POPC-POPG vesicles, the peptide transitions from a random coil towards a more structured conformation, which is even more pronounced in negatively charged SDS micelles. In vesicles, the effect depends on the peptide-lipid ratio, likely resulting from vesicle surface saturation. Further experiments with zwitterionic POPC vesicles and DPC micelles show that both curvature and negatively charged lipids can induce a change in conformation, with the two effects being cumulative. Electrostatic screening from Na+ ions reduced this effect. The oxidised form of the peptide shows a slightly lower propensity for secondary structure and retains a more random coil conformation even in the presence of PG-PC vesicles.

Investigation of Structural Peculiarities of Smooth Muscle Titin Aggregates, Formed under Different In Vitro Conditions, by Small-Angle X-Ray Scattering and Fourier Transform Infrared Spectroscopy.

Small-angle X-ray scattering (SAXS) and Fourier transform infrared (FTIR) spectroscopy were used to investigate structural peculiarities of two types of amyloid aggregates of smooth muscle titin, which differed in their morphology and ability to disaggregate, and differently bound thioflavin T dye. SAXS showed that the structure/shape of the two titin aggregate types was close to a flat shape. FTIR spectroscopy revealed no differences in the secondary structure of the two types. These data suggest that both types of "flat-shape" titin aggregates are identical in their secondary structure and, as shown previously, have a quaternary cross-ß structure. An assumption was made that the most stable supramolecular complexes of a cross-ß structure, which do not differ in their secondary structure, formed first during the aggregation of smooth muscle titin. Then, depending on ambient conditions, these supramolecular structures could form titin aggregates of different morphology and properties.

Effect of protein oxidation on the structure and emulsifying properties of fish gelatin.

This study aimed to investigate the effect of oxidation on fish gelatin and its emulsifying properties. Fish gelatin was oxidized with varying concentrations of H2O2 (0-30 mM). Increased concentrations of the oxidant led to a decrease in amino acids in the gelatin, including glycine, lysine, and arginine. Additionally, the relative content of ordered secondary structure and triple helix fractions decreased. Zeta potential decreased, while particle size, surface hydrophobicity, and water contact angle increased. Regarding emulsifying behavior, oxidation promoted the adsorption of gelatin to the oil-water interface and reduced interfacial tension. With increased degrees of oxidation, the zeta potential and size of the emulsion droplets decreased. The oxidized gelatin exhibited better emulsifying activity but worse emulsifying stability. Based on these results, a mechanism for how oxidation affects the emulsifying properties of gelatin was proposed: the increase in gelatin's hydrophobicity and the decrease in triple helix structure induced by oxidation reduced the interfacial tension at the oil-water interface. This promoted protein adsorption at the oil-water interface, allowing the formation of smaller oil droplets and enhancing gelatin's emulsifying activity. However, the decrease in electrostatic repulsion between emulsion droplets and the decrease in solution viscosity increased the flocculation and aggregation of oil droplets, ultimately weakening the emulsifying stability of gelatin.

Study of Insulin Aggregation and Fibril Structure under Different Environmental Conditions.

Protein amyloid aggregation is linked with widespread and fatal neurodegenerative disorders as well as several amyloidoses. Insulin, a small polypeptide hormone, is associated with injection-site amyloidosis and is a popular model protein for in vitro studies of amyloid aggregation processes as well as in the search for potential anti-amyloid compounds. Despite hundreds of studies conducted with this specific protein, the procedures used have employed a vast array of different means of achieving fibril formation. These conditions include the use of different solution components, pH values, ionic strengths, and other additives. In turn, this variety of conditions results in the generation of fibrils with different structures, morphologies and stabilities, which severely limits the possibility of cross-study comparisons as well as result interpretations. In this work, we examine the condition-structure relationship of insulin amyloid aggregation under a range of commonly used pH and ionic strength conditions as well as solution components. We demonstrate the correlation between the reaction solution properties and the resulting aggregation kinetic parameters, aggregate secondary structures, morphologies, stabilities and dye-binding modes.

Impact of Multi-Factor Features on Protein Secondary Structure Prediction.

Protein secondary structure prediction (PSSP) plays a crucial role in resolving protein functions and properties. Significant progress has been made in this field in recent years, and the use of a variety of protein-related features, including amino acid sequences, position-specific score matrices (PSSM), amino acid properties, and secondary structure trend factors, to improve prediction accuracy is an important technical route for it. However, a comprehensive evaluation of the impact of these factor features in secondary structure prediction is lacking in the current work. This study quantitatively analyzes the impact of several major factors on secondary structure prediction models using a more explanatory four-class machine learning approach. The applicability of each factor in the different types of methods, the extent to which the different methods work on each factor, and the evaluation of the effect of multi-factor combinations are explored in detail. Through experiments and analyses, it was found that PSSM performs best in methods with strong high-dimensional features and complex feature extraction capabilities, while amino acid sequences, although performing poorly overall, perform relatively well in methods with strong linear processing capabilities. Also, the combination of amino acid properties and trend factors significantly improved the prediction performance. This study provides empirical evidence for future researchers to optimize multi-factor feature combinations and apply them to protein secondary structure prediction models, which is beneficial in further optimizing the use of these factors to enhance the performance of protein secondary structure prediction models.

Acidocin A and Acidocin 8912 Belong to a Distinct Subfamily of Class II Bacteriocins with a Broad Spectrum of Antimicrobial Activity.

Within class II bacteriocins, we assume the presence of a separate subfamily of antimicrobial peptides possessing a broad spectrum of antimicrobial activity. Although these peptides are structurally related to the subclass IIa (pediocin-like) bacteriocins, they have significant differences in biological activities and, probably, a mechanism of their antimicrobial action. A representative of this subfamily is acidocin A from Lactobacillus acidophilus TK9201. We discovered the similarity between acidocin A and acidocin 8912 from Lactobacillus acidophilus TK8912 when analyzing plasmids from lactic acid bacteria and suggested the presence of a single evolutionary predecessor of these peptides. We obtained the C-terminally extended homolog of acidocin 8912, named acidocin 8912A, a possible intermediate form in the evolution of the former. The study of secondary structures and biological activities of these peptides showed their structural similarity to acidocin A; however, the antimicrobial activities of acidocin 8912 and acidocin 8912A were lower than that of acidocin A. In addition, these peptides demonstrated stronger cytotoxic and membranotropic effects. Building upon what we previously discovered about the immunomodulatory properties of acidocin A, we studied its proteolytic stability under conditions simulating those in the digestive tract and also assessed its ability to permeate intestinal epithelium using the Caco-2 cells monolayer model. In addition, we found a pronounced effect of acidocin A against fungi of the genus Candida, which might also expand the therapeutic potential of this bacterial antimicrobial peptide.

Secondary structure changes as the potential H2 sensing mechanism of group D [FeFe]-hydrogenases.

[FeFe]-hydrogenases function as both H2 catalysts and sensors. While catalysis is well investigated, details regarding the H2 sensing mechanism are limited. Here, we relate protein structure changes to H2 sensing, similar to light-driven bio-sensors. Our results highlight how identical cofactors incorporated in alternative protein scaffolds serve different functions in nature.

A structural rationale for reversible vs irreversible amyloid fibril formation from a single protein.

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-ß motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-ß-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of ß-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a "memory" of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-ß structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils.

Static magnetic field effects on the secondary structure and elasticity of collagen molecules; a possible biophysical approach to treat keratoconus.

Type I collagen is among the major extracellular proteins that play a significant role in the maintenance of the cornea's structural integrity and is essential in cell adhesion, differentiation, growth, and integrity. Here, we investigated the effect of 300 mT Static Magnetic Field (300 mT SMF) on the structure and molecular properties of acid-solubilized collagens (ASC) isolated from the rat tail tendon. The SMF effects at molecular and atomic levels were investigated by various biophysical approaches like Circular Dichroism Spectropolarimetery (CD), Fourier Transform Infrared Spectroscopy (FTIR), Zetasizer light Scattering, and Rheological assay. Exposure of isolated type I collagen to 300 mT SMF retained its triple helix. The elasticity of collagen molecules and the keratoconus (KCN) cornea treated with SMF decreased significantly after 5 min and slightly after 10, 15, and 20 min of treatments. The exposure to 300 mT SMF shifted the Amid I bond random coil to antiparallel wave number from 1647 to 1631 cm-1. The pH of the 300 mT SMF treated collagen solution increased by about 25 %. The treatment of the KCN corneas with 300 mT SMF decreased their elasticity significantly. The promising results of the effects of 300 mT SMF on the collagen molecules and KCN cornea propose a novel biophysical approach capable of manipulating the collagen's elasticity, surface charges, electrostatic interactions, cross binding, network formation and fine structure. Therefore, SMF treatment may be considered as a novel non-invasive, direct, non-chemical and fast therapeutic and manipulative means to treat KCN cornea where the deviated physico-chemical status of collagen molecules cause deformation.

Protonation State-Induced Unfolding of Protein Secondary Structure in the Gas Phase.

The combination of infrared spectroscopy (IR) and ion mobility mass spectrometry (IM-MS) has revealed that protein secondary structures are retained upon transformation from aqueous solution to the gas phase under gentle conditions. Yet the details about where and how these structural elements are embedded in the gas phase remain elusive. In this study, we employ long time scale molecular dynamics (MD) simulations to examine the extent to which proteins retain their solution structures and the impact of protonation state on the stability of secondary structures in the gas phase. Our investigation focuses on two well-studied proteins, myoglobin and ß-lactoglobulin, representing typical helical and ß-sheet proteins, respectively. Our simulations accurately reproduce the experimental collision cross section (CCS) data measured by IM-MS. Based on accurately reproducing previous experimental collision cross section data and dominant secondary structural species obtained from IM-MS and IR, we confirm that both proteins largely retain their native secondary structural components upon passing from aqueous solution to the gas phase. However, we observe significant reductions in secondary structure contents (19.2 ± 1.2% for myoglobin and 7.3 ± 0.6% for ß-lactoglobulin) in specific regions predominantly composed of ionizable residues. Further mechanistic analysis suggests that alterations in protonation states of these residues after phase transition induce changes in their local interaction networks and backbone dihedral angles, which potentially promote the unfolding of secondary structures in the gas phase. We anticipate that similar protonation state induced unfolding may be observed in other proteins possessing distinct secondary structures. Further studies on a broader array of proteins will be essential to refine our understanding of protein structural behavior during the transition to the gas phase.

Cryo-EM and solid state NMR together provide a more comprehensive structural investigation of protein fibrils.

The tropomyosin 1 isoform I/C C-terminal domain (Tm1-LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo-EM) and solid state nuclear magnetic resonance (NMR). This study demonstrates the complementary nature of these two structural biology techniques. Chemical shift assignments from solid state NMR are used to determine the secondary structure at the level of individual amino acids, which is faithfully seen in cryo-EM reconstructions. Additionally, solid state NMR demonstrates that the region not observed in the reconstructed cryo-EM density is primarily in a highly mobile random coil conformation rather than adopting multiple rigid conformations. Overall, this study illustrates the benefit of investigations combining cryo-EM and solid state NMR to investigate protein fibril structure.

Rapid Quantification of Protein Secondary Structure Composition from a Single Unassigned 1D 13C Nuclear Magnetic Resonance Spectrum.

The function of a protein is predicated upon its three-dimensional fold. Representing its complex structure as a series of repeating secondary structural elements is one of the most useful ways by which we study, characterize, and visualize a protein. Consequently, experimental methods that quantify the secondary structure content allow us to connect a protein's structure to its function. Here, we introduce an automated gradient descent-based method we refer to as secondary-structure distribution by NMR that allows for rapid quantification of the protein secondary structure composition of a protein from a single, 1D 13C NMR spectrum without chemical shift assignments. The analysis of nearly 900 proteins with known structure and chemical shifts demonstrates the capabilities of our approach. We show that these results rival alternative techniques such as FT-IR and circular dichroism that are commonly used to estimate secondary structure compositions. The resulting method requires only the primary sequence of the protein and its referenced 13C NMR spectrum. Each residue is modeled in an ensemble of secondary structures with percentage contributions from random coil, α-helix, and ß-sheet secondary structures obtained by minimizing the difference between a simulated and experimental 1D 13C NMR spectrum. The capabilities of the method are demonstrated as applied to samples at natural abundance or enriched in 13C, acquired by either solution or solid-state NMR, and even on low magnetic field benchtop NMR spectrometers. This approach allows for rapid characterization of protein secondary structure across traditionally challenging to characterize states including liquid-liquid phase-separated, membrane-bound, or aggregated states.

Structure Derivatization of IgG-Binding Peptides and Analysis of Their Secondary Structure by Circular Dichroism Spectroscopy.

Mid-sized cyclic peptides are a promising modality for modern drug discovery. Their larger interaction area coupled with an appropriate secondary structure is more suitable than small molecules for binding to the target protein. In this study, we conducted a structure derivatization of an immunoglobulin G (IgG)-binding peptide (15-IgBP), a ß-hairpin-like cyclic peptide with a twisted ß-strand and assessed the effect of the secondary structure on IgG-binding activity using circular dichroism (CD) spectra analysis. As a result, derivatization at the Ala5 and Gly9 positions affected the secondary structure of 15-IgBP, in particular the appearance of a small positive peak in the 220-240 nm region characteristic of 15-IgBP in the CD spectrum. Maintaining this peak at a moderate level may be important for the expression of IgG binding activity. We found the small methyl group at Ala5 to be crucial for retaining the preferred secondary structure; we also found Gly9 could be replaced by D-amino acids. By integrating these findings with previous results of the structure-activity relationship, we obtained four potent affinity peptides for IgG binding (Kd = 4.24-5.85 nM). Furthermore, we found the Gly9 position can be substituted for D-Lys. This is a new potential site for attaching functional units for conjugation with IgG for the preparation of homogeneous antibody-drug conjugates.

bio2Byte Tools deployment as a Python package and Galaxy tool to predict protein biophysical properties.

SUMMARY: We introduce a unified Python package for the prediction of protein biophysical properties, streamlining previous tools developed by the Bio2Byte research group. This suite facilitates comprehensive assessments of protein characteristics, incorporating predictors for backbone and sidechain dynamics, local secondary structure propensities, early folding, long disorder, beta-sheet aggregation, and fused in sarcoma (FUS)-like phase separation. Our package significantly eases the integration and execution of these tools, enhancing accessibility for both computational and experimental researchers. AVAILABILITY AND IMPLEMENTATION: The suite is available on the Python Package Index (PyPI): https://pypi.org/project/b2bTools/ and Bioconda: https://bioconda.github.io/recipes/b2btools/README.html for Linux and macOS systems, with Docker images hosted on Biocontainers: https://quay.io/repository/biocontainers/b2btools?tab=tags&tag=latest and Docker Hub: https://hub.docker.com/u/bio2byte. Online deployments are available on Galaxy Europe: https://usegalaxy.eu/root?tool_id=b2btools_single_sequence and our online server: https://bio2byte.be/b2btools/. The source code can be found at https://bitbucket.org/bio2byte/b2btools_releases.

Enumerative Discovery of Noncanonical Polypeptide Secondary Structures.

Energetically favorable local interactions can overcome the entropic cost of chain ordering and cause otherwise flexible polymers to adopt regularly repeating backbone conformations. A prominent example is the α helix present in many protein structures, which is stabilized by i, i + 4 hydrogen bonds between backbone peptide units. With the increased chemical diversity offered by unnatural amino acids and backbones, it has been possible to identify regularly repeating structures not present in proteins, but to date, there has been no systematic approach for identifying new polymers likely to have such structures despite their considerable potential for molecular engineering. Here we describe a systematic approach to search through dipeptide combinations of 130 chemically diverse amino acids to identify those predicted to populate unique low-energy states. We characterize ten newly identified dipeptide repeating structures using circular dichroism spectroscopy and comparison with calculated spectra. NMR and X-ray crystallographic structures of two of these dipeptide-repeat polymers are similar to the computational models. Our approach is readily generalizable to identify low-energy repeating structures for a wide variety of polymers, and our ordered dipeptide repeats provide new building blocks for molecular engineering.

Geometric descriptors for beta turns.

Beta turns, in which the protein backbone abruptly changes direction over four amino acid residues, are the most common type of protein secondary structure after alpha helices and beta sheets and play key structural and functional roles. Previous work has produced classification systems for turn geometry at multiple levels of precision, but these operate in backbone dihedral-angle (Ramachandran) space, and the absence of a local Euclidean-space coordinate system and structural alignment for turns, or of any systematic Euclidean-space characterization of turn backbone shape, presents challenges for the visualization, comparison and analysis of the wide range of turn conformations and the design of turns and the structures that incorporate them. This work derives a turn-local coordinate system that implicitly aligns turns, together with a set of geometric descriptors that characterize the bulk BB shapes of turns and describe modes of structural variation not explicitly captured by existing systems. These modes are shown to be meaningful by the demonstration of clear relationships between descriptor values and the electrostatic energy of the beta-turn H-bond, the overrepresentations of key side-chain motifs, and the structural contexts of turns. Geometric turn descriptors complement Ramachandran-space classifications, and they can be used to select turn structures for compatibility with particular side-chain interactions or contexts. Potential applications include protein design and other tasks in which an enhanced Euclidean-space characterization of turns may improve understanding or performance. The web-based tools ExploreTurns, MapTurns, and ProfileTurn, available at www.betaturn.com, incorporate turn-local coordinates and turn descriptors and demonstrate their utility.

Insight into the effects of large yellow croaker roe (Larimichthys Crocea) phospholipids on the conformational and functional properties of pork myofibrillar protein.

The large yellow croaker roe phospholipids (LYPLs), rich in polyunsaturated fatty acids, is a potential phospholipid additive for meat products. In this work, the effects of LYPLs on the structural and functional properties of myofibrillar protein (MP) were determined, and compared with egg yolk phospholipids (EYPLs) and soybean phospholipids (SBPLs). The results revealed that LYPLs, similar to SBPLs and EYPLs, induced a transformation in the secondary structure of MP from α-helix to ß-sheets and random coils, while also inhibited the formation of carbonyl and disulfide bonds within MP. All three phospholipids induced MP tertiary structure unfolding, with the greatest degree of unfolding observed in MP containing LYPLs. The MP with LYPLs had the highest surface hydrophobicity, emulsification properties and gel strength. In addition, MP with LYPLs added also demonstrated superior rheological properties and water-holding capacity compared with SBPLs and EYPLs. In conclusion, adding LYPLs endowed MP with improved functional properties.