Protein Functionality Research Articles

Engineering of protein glutaminase for highly efficient modification of fish myofibrillar protein through structure-based and computational-aided strategy

Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp31, Ser72, Asn121, Asp471, and Glu485 were crucial for maintaining PG5-myosin interaction, with Ser72 and Asn121 making significant energy contributions.

Effect of ultrafiltration of acid whey at pH 4.6 or 6.5 on whey concentrate composition and resultant powder properties

This study investigated the effect of modifying the mineral composition through adjustment of pH prior to ultrafiltration on the functionality of acid whey protein concentrate (WPC; pH 4.6). Increasing pH to 6.5 prior to ultrafiltration produced a concentrate with a significantly higher calcium and phosphorous content, compared to ultrafiltration at pH 4.6. WPC produced at pH 6.5 also had significantly more calcium‐phosphate precipitation following pasteurisation and evaporation and had a higher viscosity at 30% solids (300 s−1, 50°C). In addition, both reconstituted powders (5%, w/w, protein) had strong gelation properties following heat treatment at 90°C; however, the WPC produced at pH 6.5 had a significantly firmer gel strength, but such gels appeared to be more sensitive to breakage at increased frequency and oscillation strain. Overall, the pH prior to UF had a significant impact on the processing of acid whey and final product functionality.

Post-translational modification prediction via prompt-based fine-tuning of a GPT-2 model

Post-translational modifications (PTMs) are pivotal in modulating protein functions and influencing cellular processes like signaling, localization, and degradation. The complexity of these biological interactions necessitates efficient predictive methodologies. In this work, we introduce PTMGPT2, an interpretable protein language model that utilizes prompt-based fine-tuning to improve its accuracy in precisely predicting PTMs. Drawing inspiration from recent advancements in GPT-based architectures, PTMGPT2 adopts unsupervised learning to identify PTMs. It utilizes a custom prompt to guide the model through the subtle linguistic patterns encoded in amino acid sequences, generating tokens indicative of PTM sites. To provide interpretability, we visualize attention profiles from the model’s final decoder layer to elucidate sequence motifs essential for molecular recognition and analyze the effects of mutations at or near PTM sites to offer deeper insights into protein functionality. Comparative assessments reveal that PTMGPT2 outperforms existing methods across 19 PTM types, underscoring its potential in identifying disease associations and drug targets.

Pulsed Electric Fields Effects on Proteins: Extraction, Structural Modification, and Enhancing Enzymatic Activity.

Pulsed electric field (PEF) is an innovative physical method for food processing characterized by low energy consumption and short processing time. This technology represents a sustainable procedure to extend food shelf-life, enhance mass transfer, or modify food structure. The main mechanism of action of PEF for food processing is the increment of the permeability of the cell membranes by electroporation. However, it has also been shown that PEF may modify the technological and functional properties of proteins. Generating a high-intensity electric field necessitates the flow of an electric current that may have side effects such as electrochemical reactions and temperature increments due to the Joule effect that may affect food components such as proteins. This article presents a critical review of the knowledge on the extraction of proteins assisted by PEF and the impact of these treatments on protein composition, structure, and functionality. The required research for understanding what happens to a protein when it is under the action of a high-intensity electric field and to know if the mechanism of action of PEF on proteins is different from thermal or electrochemical effects is underlying.

Molecular Insights into the Binding and Conformational Changes of Hepcidin25 Blood Peptide with 4-Aminoantipyrine and Their Sorption Mechanism by Carboxylic-Functionalized Multiwalled Carbon Nanotubes: A Comprehensive Spectral Analysis and Molecular Dynamics Simulation Study.

In this work, the main purpose is to analyze and understand the mechanism and thermodynamic interactions of carboxylic acid-functionalized multiwalled carbon nanotubes (cf-MWCNTs) and 4-aminoantipyrine (AAP) with human hepcidine25 (Hep25) using multispectroscopic and molecular docking modeling methods, binding free energy calculations, and molecular dynamics (MD) simulations under physiological conditions. AAP belongs to a class of persistent environmental contaminants, and its residue is a potential hazard to human health, exhibiting a high binding affinity with blood peptides. Hepcidin is a 25-residue peptide hormone with four disulfide bonds that regulates the iron balance in vertebrates and contributes to host immunity as a cysteine-rich antimicrobial peptide. Due to their diverse properties and pollutant absorption capabilities, CNTs demonstrate important biological effects in biological applications, particularly in the noncovalent interactions with blood peptides. A comprehensive molecular dynamics simulation integrated with molecular docking methodologies was employed to explore the binding free energy between AAP and Hep25, identify binding sites, elucidate thermodynamic characteristics, and evaluate the binding forces governing their interaction. The investigation delved into elucidating the precise binding site of AAP within the Hep25 protein and thoroughly analyzed the impact of AAP on the microenvironment and conformational dynamics of Hep25. The circular dichroism (CD) experimental results highlight a reduction in β-sheet composition following the introduction of AAP and cf-MWCNT. In addition, outcomes from fluorescence spectroscopy demonstrate that both cf-MWCNT and AAP significantly attenuated Hep-25 fluorescence via a static quenching mechanism. According to the MD simulations, the presence of AAP induces changes in the secondary structure of Hep25 and enhances its hydrophobicity. Additionally, our findings demonstrated that alongside the alteration in protein structure and functionality induced by contaminants, cf-MWCNTs possess the capability to mitigate the contaminant-induced effects on Hep25 activity while preserving the overarching structural integrity of Hep25. Based on the distance and RDF data, we found that during the simulation the presence of the cf-MWCNT causes the AAP to move away from the Hep25, and as a result fewer and weaker interactions of the AAP with the Hep25 will be observed. Likewise, free energy calculations indicate that the binding of Hep25 to AAP and cf-MWCNT involves electrostatic, π-cationic, and π-π stacking interactions. The research findings offer invaluable insights into the intricate influence of pollutants and carbon nanotubes on protein functionality within the circulatory system and their toxicity in vivo for prospective investigations.

Characterization of a Novel Pathogenic PLCG2 Variant Leading to APLAIDSyndrome Responsive to a TNF Inhibitor.

Autoinflammation and phospholipase C (PLC) γ2-associated antibody deficiency and immune dysregulation (APLAID) syndrome is an autoinflammatory disease caused by gain-of-function variants in PLCG2. This study investigates the pathogenic mechanism of a novel variant of PLCG2 in a patient with APLAID syndrome. Whole-exome sequencing and Sanger sequencing were used to identify the pathogenic variant in the patient. Single-cell RNA sequencing, immunoblotting, luciferase assay, inositol monophosphate enzyme-linked immunosorbent assay, calcium flux assay, quantitative PCR, and immunoprecipitation were used to define inflammatory signatures and evaluate the effects of the PLCG2 variant on protein functionality and immune signaling. We identified a novel de novo variant, PLCG2 p.D993Y, in a patient with colitis, pansinusitis, skin rash, edema, recurrent respiratory infections, B-cell deficiencies, and hypogammaglobulinemia. The single-cell transcriptome revealed exacerbated inflammatory responses in the patient's peripheral blood mononuclear cells. Expression of the D993Y variant in HEK293T, COS-7, and PLCG2 knock-out THP-1 cell lines showed heightened PLCγ2 phosphorylation; elevated inositol-1,4,5-trisphosphate production and intracellular Ca2+ release; and activation of the MAPK, NF-κB, and NFAT signaling pathways compared with control-transfected cells. In vitro experiments indicated that the D993Y variant altered amino acid properties, disrupting the interaction between the catalytic and autoinhibitory domains of PLCγ2, resulting in PLCγ2 autoactivation. Our findings demonstrated that the PLCG2 D993Y variant is a gain-of-function mutation via impairing its autoinhibition, activating multiple inflammatory signaling pathways, thus leading to APLAID syndrome. This study further broadens the molecular underpinnings and phenotypic spectrum of PLCγ2-related disorders.

Breaking the Degeneracy of Sense Codons – How Far Can We Go?

AbstractGenetic code expansion aims to incorporate non‐canonical amino acids (ncAAs) into biological systems, enhancing protein functionality or enabling the in vitro selection of peptides from diverse mRNA displayed libraries. Typically, genetic code expansion has involved reassignment of stop codons to ncAAs through orthogonal translation systems. This review instead focuses on efforts to expand the genetic code by breaking the redundancy of sense codons in vitro and in vivo. In vivo, orthogonal aminoacyl‐tRNA synthetase (AARS)/tRNA/AA systems are able to compete with endogenous machinery, enabling partial to full codon reassignment. Recent approaches, like genome recoding, offer potential solutions to reduce competition. In vitro studies utilize cell extract‐based or reconstituted translation systems, allowing precise control of codon usage via gene design and tRNA addition, making breaking of sense degeneracy easier. In these systems several unsplit codon boxes have been successfully reassigned multiple to ncAAs. These efforts showcase both the successes and challenges in achieving orthogonality and selective codon decoding and point towards a future where the 64 codons can encode more than 30 monomers, enabling new advances in synthetic biology and drug discovery.

Accurate prediction of protein function using statistics-informed graph networks

Understanding protein function is pivotal in comprehending the intricate mechanisms that underlie many crucial biological activities, with far-reaching implications in the fields of medicine, biotechnology, and drug development. However, more than 200 million proteins remain uncharacterized, and computational efforts heavily rely on protein structural information to predict annotations of varying quality. Here, we present a method that utilizes statistics-informed graph networks to predict protein functions solely from its sequence. Our method inherently characterizes evolutionary signatures, allowing for a quantitative assessment of the significance of residues that carry out specific functions. PhiGnet not only demonstrates superior performance compared to alternative approaches but also narrows the sequence-function gap, even in the absence of structural information. Our findings indicate that applying deep learning to evolutionary data can highlight functional sites at the residue level, providing valuable support for interpreting both existing properties and new functionalities of proteins in research and biomedicine.

How pulse electric field treatment affects anti-nutritional factors and plant protein digestibility: A concise review

Consumption of foods rich in anti-nutritional factors (ANFs) can lead to inadequate absorption of vitamins and minerals. Pulsed electric field (PEF) treatment is a non-thermal food processing technology that uses short bursts of high voltage electric fields to induce structural modifications in biomolecules, including proteins, through electrochemical reactions and polarization of structural moieties. PEF offers potential benefits in enhancing the nutritional quality of food products by altering the structure and function of ANFs. However, the effects of PEF on ANFs in food have not been well established. Additionally, PEF-treated samples may undergo protein structural orientation modification due to electroporation and free radical species, potentially unfolding the native structure and influencing protein digestibility. Moreover, by increasing the mass transfer rate, electroporation can enhance the extraction of the initially trapped fractions into the solvent medium, which may include target compounds that affect protein functionality and digestibility. However, the protein digestibility of PEF-treated food products is yet to be thoroughly explored. Therefore, this review objectively investigates the possible mechanisms involved and the effectiveness of PEF in reducing different ANFs and modifying plant protein digestibility. This review also provides in-depth information about the possible applications of PEF-treated food items in the food industry.

Influence of hydrothermal processing conditions on the functionality of Phaeodactylum tricornutum extracts

Microalgae are an underutilized biomass source of nutritionally valuable extracts with promising texturizing capacities that can be used for food applications. This also requires the development of environmentally friendly extraction technologies. Hydrothermal processing was used in this study to investigate the effects of temperature (up to 225 °C) on the extracts from the diatom Phaeodactylum tricornutum. The relationship between protein and polysaccharides' presence and functionality was assessed, based on structure, composition, bioactivity, and rheological behavior. An optimal condition of non-isothermal processing at 120 °C resulted in the extract with gelling capacity (at 20 % w/w extract concentration). This extract is rich in carbohydrates in oligosaccharide form (9 g per 100 g extract) and protein (16 g per 100 g extract), which indicates nutritional relevance and possible prebiotic effects. Moreover, after an additional purification step with food-grade solvents, this extract was able to gel at 5 % w/w concentration. These combined with its significant antioxidant activity (over 100 μmol Trolox equivalents per gram of extract in all studied conditions, using the ABTS method) point towards new possibilities for the application of Phaeodactylum tricornutum extracts in novel clean-label vegan foods. Overall, these findings suggest that the novel approach proposed in this work, based on hydrothermal processing, is feasible and compatible with the need for the application of simple purification steps with food-grade solvents. Furthermore, it reinforced the relevance of the carbohydrate fractions and carbohydrate-protein interactions from microalgae.

Synthesis of Polypeptides by Ring-opening Polymerization: A Concise Review

Abstract: The most economical and efficient route to prepare polypeptides from synthetic chemistry is through the Ring-opening Polymerization (ROP) of amino acids using Ncarboxyanhydride (NCA) monomers. Peptide polymers, in contrast to proteins, consist of repeated amino acid units and are comparatively simpler macromolecules. Despite their simplicity, these polypeptides offer a unique combination of beneficial traits found in both synthetic polymers (such as solubility, processability, and rubber elasticity) and natural proteins (including secondary structure, functionality, and biocompatibility). Nevertheless, NCA polymerization faces significant challenges, including intricate monomer purification and the necessity for processing toxic solvents. In this context, this review presents the fundamental principles of this polymer chemistry, the synthesis of NCA monomers, and the different methodologies to access polypeptides by ROP. It also explores the most recent advances in this field of research, with a focus on how new methods enable the use of more reactive initiators and the development of original processes, including the use of aqueous solvents.

Unraveling the impact of deleterious nsSNPs on the MFSD1 protein

MFSD1 (Major facilitator superfamily domain containing 1) protein is a lysosomal multiple transmembrane-spanning protein responsible for the active transport of various compounds. Due to its potential role in maintaining liver homeostasis, any mutation might lead to altered protein expression, thus affecting the functionality. In this study, we explored the impact of deleterious nsSNPs (Nonsynonymous single nucleotide polymorphisms) on the stability, conformation, and functionality of the MFSD1 protein. SNP data and MFSD1 protein sequences were retrieved from NCBI dbSNP and Uniprot respectively. In total, five highly conserved nsSNPs were predicted to be deleterious based on their negative impact on the stability of the protein. Furthermore, the simulation analysis on the 3D structures of both native and mutant proteins revealed a notable impact on the physiological conformation of the protein. The identified variants not only affect the native conformation but also impact the association of MFSD1 with GLMP (Glycosylated Lysosomal Membrane Protein). In conclusion, the analysis revealed that the mutant protein's structural stability was inferior to that of the native protein. This research provides crucial insights for identifying and assessing MFSD1 mutations as potential diagnostic markers for liver-related diseases.

Semi-Covariance Coefficient Analysis of Spike Proteins from SARS-CoV-2 and Its Variants Omicron, BA.5, EG.5, and JN.1 for Viral Infectivity, Virulence and Immune Escape.

Semi-covariance has attracted significant attention in recent years and is increasingly employed to elucidate statistical phenomena exhibiting fluctuations, such as the similarity or difference in charge patterns of spike proteins among coronaviruses. In this study, by examining values above and below the average/mean based on the positive and negative charge patterns of amino acid residues in the spike proteins of SARS-CoV-2 and its current circulating variants, the proposed methods offer profound insights into the nonlinear evolving trends in those viral spike proteins. Our study indicates that the charge span value can predict the infectivity of the virus and the charge density can estimate the virulence of the virus, and both predicated infectivity and virulence appear to be associated with the capability of viral immune escape. This semi-covariance coefficient analysis may be used not only to predict the infectivity, virulence and capability of immune escape for coronaviruses but also to analyze the functionality of other viral proteins. This study improves our understanding of the trend of viral evolution in terms of viral infectivity, virulence or the capability of immune escape, which remains further validated by more future studies and statistical data.

Exome sequencing reveals pathogenic mutations in the LARS2 and HSD17B4 genes associated with Perrault syndrome and D-bifunctional protein deficiency in Moroccan families.

Syndromic hearing loss (SHL) is characterized by hearing impairment accompanied by other clinical manifestations, reaching over 400 syndromes. Early and accurate diagnosis is essential to understand the progression of hearing loss and associated systemic complications. In this study, we investigated the genetic etiology of sensorineural hearing loss in three Moroccan patients using whole exome sequencing (WES). The results revealed in two families Perrault syndrome caused by LARS2, p. Asn153His; p. Thr629Met compound heterozygous variants in two siblings in one family; and p. Thr522Asn, a homozygous variant in two sisters in another. The patient in the third family was diagnosed with D-bifunctional protein deficiency (D-BPD), linked to compound heterozygous mutations p. Asn457Tyr and p. Val643Argfs*5 in HSD17B4. Molecular dynamic simulation results showed that Val643Argfs*5 does not prevent HSD17B4 protein from binding to the PEX5 receptor, but further studies are recommended to verify its effect on HSD17B4 protein functionality. These results highlight the effectiveness of WES in identifying pathogenic mutations involved in heterogeneous disorders and the usefulness of bioinformatics in predicting their effects on protein structure.

Decoding the Role of O-GlcNAcylation in Hepatocellular Carcinoma.

Post-translational modifications (PTMs) influence protein functionality by modulating protein stability, localization, and interactions with other molecules, thereby controlling various cellular processes. Common PTMs include phosphorylation, acetylation, ubiquitination, glycosylation, SUMOylation, methylation, sulfation, and nitrosylation. Among these modifications, O-GlcNAcylation has been shown to play a critical role in cancer development and progression, especially in hepatocellular carcinoma (HCC). This review outlines the role of O-GlcNAcylation in the development and progression of HCC. Moreover, we delve into the underlying mechanisms of O-GlcNAcylation in HCC and highlight compounds that target O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) to improve treatment outcomes. Understanding the role of O-GlcNAcylation in HCC will offer insights into potential therapeutic strategies targeting OGT and OGA, which could improve treatment for patients with HCC.

Photoswitchable Auxin-Inducible Degron System for Conditional Protein Degradation with Spatiotemporal Resolution.

The auxin-inducible degron (AID) system degrades target proteins rapidly in a controllable manner. Although this is a highly versatile technique for studying protein functionality, protein degradation with spatiotemporal resolution is not currently possible. Herein we describe a photoswitchable AID using a light-active auxin derivative for reversible and site-specific protein degradation with temporal resolution.

Structure, emulsifying and embedding characteristics of soy protein isolate induced by the complexation of carrageenan with different charge groups

Soy protein isolate (SPI) offers high protein, nutritional benefits, and versatile functionality but requires modification due to its environmental sensitivity and functional limitations. To expand the functional characteristics and application scope of SPI, three types of carrageenan (Car) was used to form complexes with SPI. With the addition of carrageenan, the turbidity of SPI at its isoelectric point significantly decreased, and the turbidity curve shifted toward the acidic range. Structural analysis showed that λ-carrageenan significantly reduced the hydrophobicity of SPI from 218 to 150 when combined to SPI at a Car/SPI ratio of 1:5 (P < 0.05). Fourier transform infrared spectra revealed that the electrostatic interaction between ι-carrageenan and SPI was the strongest at a ratio of 1:5. The emulsion stabilized by κ-Car/SPI complexes had the smallest creaming index, with a creaming index of 46.6% after 7 days of storage, which was 33.3% lower than the creaming index of the SPI emulsion (P < 0.05). The ι-Car/SPI complexes exhibited the highest encapsulation efficiency for curcumin, with a value of 80.1%, which was 25.3% higher than the SPI (P < 0.05). This study could provide solutions for the modification of SPI and enhance its application in the food industry.

Rapid and Efficient Isolation of Total RNA-Bound Proteomes by Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP).

The critical roles of RNA-binding proteins (RBPs) in all aspects of RNA biology fostered the development of methods utilizing ultraviolet (UV) crosslinking and method-specific RNA enrichment steps for proteome-wide identification and assessment of RBP function. Despite the substantial contributions of these UV-based RNA-centric methods to our understanding of RNA-protein interaction networks, their utility is constrained by biases in RBP recovery and significant noise contributions, which can confound meaningful interpretation. To overcome these issues, we recently developed a method termed Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP) and introduced quantitative signal-to-noise (S:N)-based metrics for the proteome-wide identification of RNA interactomes and accurate assessment of global RBP occupancy dynamics. Compared to existing methodologies, LEAP-RBP provides significant advantages in speed, cost, efficiency, and selectivity for RNA-bound proteins. In this work, we provide a step-by-step guide for the successful application of the LEAP-RBP method for both small- and large-scale investigations of RNA-bound proteomes. Key features • Unbiased and efficient isolation of total RNA-bound protein, RNA, and protein from biological samples. • Cost-effective identification of proteome-wide RNA interactomes and validation of direct RNA-binding protein functionality. • Robust and accurate assessment of context- and/or condition-dependent RBP occupancy state dynamics.

Enhancing Meat Product Quality: Exploring the Effects of Additives on Myofibrillar Protein Functionality

Enhancing Meat Product Quality: Exploring the Effects of Additives on Myofibrillar Protein Functionality

Ingredient Functionality of Soy, Chickpea, and Pea Protein before and after Dry Heat Pretreatment and Low Moisture Extrusion.

This study investigates the impact of dry heat pretreatment on the functionality of soy, chickpea, and pea protein ingredients for use in texturized vegetable protein (TVP) production via low moisture extrusion. The protein powders were heat-treated at temperatures ranging from 80 °C to 160 °C to modulate the extent of protein denaturation and assess their effects on RVA pasting behavior, water absorption capacity (WAC), and color attributes. The results indicate that the pretreatment temperature significantly influenced the proteins' functional properties, with an optimal temperature of 120 °C enhancing pasting properties and maintaining WAC, while a higher pretreatment temperature of 160 °C led to diminished ingredient functionality. Different protein sources exhibited distinct responses to heat pretreatment. The subsequent extrusion processing revealed significant changes in extrudate density and color, with increased density and darkness observed at higher pretreatment temperatures. This research provides insights into the interplay between protein sources, pretreatment conditions, and extrusion outcomes, highlighting the importance of controlled protein denaturation for developing high-quality, plant-based meat analogues. The findings have broad implications for the optimization of meat analogue manufacturing, with the aim of enhancing the sensory experience and sustainability of plant-based foods.