Secondary Structure Properties Research Articles

Effects of different ohmic heating treatments on parvalbumin structure and reduction of allergenicity in Japanese eel (Anguilla japonica)

We investigated the effects of ohmic heating (OH) on the structural properties and allergenicity of parvalbumin (PV). Compared to other heating methods (water bath heating (WH), OH combined with WH, and OH combined with air thermostatic heating (AH)), pure OH heating expended the least time and total energy. PV sensitization was reduced by approximately 65% by pure OH heating. SDS-PAGE, tricine–SDS-PAGE, and western blotting analyses revealed a molecular weight of sensitized β-PV of about 12 kDa. Band intensity decreased with increasing OH time, and significant changes were observed in amino acid content, secondary structure, microstructure, and dielectric properties. Reducing PV, allergenicity through protein unfolding and secondary structural changes, thereby possibly reducing the allergenicity of eel, provides a theoretical basis for developing hypoallergenic products.

Isolation and Characterization of Protein Fractions for Valorization of Sacha Inchi Oil Press-Cake.

The growing interest in plant-based food protein sources has provided opportunities for the valorization of agri-food by-products, driving the food industry towards more sustainable development. In this study, three extraction procedures (varying the pH value (7.0 and 11.0) and the addition of salt (0 and 5%)) were investigated to obtain seven different protein fractions (SIPF) from Sacha Inchi oil press-cake (SIPC), which were characterized in terms of their protein content, electrophoretic profile, secondary structure, and techno-functional properties. Extractions at pH 11.0 without salt addition produced the highest values of protein content, extraction yield, protein recovery, and protein concentration increase (84.0%, 24.7%, 36.5%, and 1.5-fold, respectively). Under these extraction conditions, the electrophoretic analysis indicated that most of the SIPC proteins were extracted. SIPF displayed an excellent oil absorption capacity (4.3-9.0 w/w), and interesting foam activity (36.4-133.3%). The solubility and emulsifying activity of the albumin fractions were significantly higher than those of the other fractions (~87 vs. <15.8%, and 280-370 vs. <140 m2/g, respectively). Correlation analysis showed that the secondary structure of the SIPF significantly influences their techno-functional properties. These results indicate that SIPC is a by-product of great potential for protein extraction processes, and as a valorization strategy for technical cycle solutions for the Sacha Inchi productive chain in the circular economy context.

Insights into the material properties of dragline spider silk affecting Schwann cell migration

Dragline silk of Trichonephila spiders has attracted attention in various applications. One of the most fascinating uses of dragline silk is in nerve regeneration as a luminal filling for nerve guidance conduits. In fact, conduits filled with spider silk can measure up to autologous nerve transplantation, but the reasons behind the success of silk fibers are not yet understood.In this study dragline fibers of Trichonephila edulis were sterilized with ethanol, UV radiation, and autoclaving and the resulting material properties were characterized with regard to the silk's suitability for nerve regeneration. Rat Schwann cells (rSCs) were seeded on these silks in vitro and their migration and proliferation were investigated as an indication for the fiber's ability to support the growth of nerves. It was found that rSCs migrate faster on ethanol treated fibers. To elucidate the reasons behind this behavior, the fiber's morphology, surface chemistry, secondary protein structure, crystallinity, and mechanical properties were studied. The results demonstrate that the synergy of dragline silk's stiffness and its composition has a crucial effect on the migration of rSCs. These findings pave the way towards understanding the response of SCs to silk fibers as well as the targeted production of synthetic alternatives for regenerative medicine applications.

Amyloid fibrils degradation: the pathway to recovery or aggravation of the disease?

Background: The most obvious manifestation of amyloidoses is the accumulation of amyloid fibrils as plaques in tissues and organs, which always leads to a noticeable deterioration in the patients' condition and is the main marker of the disease. For this reason, early diagnosis of amyloidosis is difficult, and inhibition of fibrillogenesis, when mature amyloids are already accumulated in large quantities, is ineffective. A new direction for amyloidosis treatment is the development of approaches aimed at the degradation of mature amyloid fibrils. In the present work, we investigated possible consequences of amyloid's degradation. Methods: We analyzed the size and morphology of amyloid degradation products by transmission and confocal laser scanning microscopy, their secondary structure and spectral properties of aromatic amino acids, intrinsic chromophore sfGFP, and fibril-bound amyloid-specific probe thioflavin T (ThT) by the absorption, fluorescence and circular dichroism spectroscopy, as well as the cytotoxicity of the formed protein aggregates by MTT-test and their resistance to ionic detergents and boiling by SDS-PAGE. Results: On the example of sfGFP fibrils (model fibrils, structural rearrangements of which can be detected by a specific change in the spectral properties of their chromophore), and pathological Aβ-peptide (Aβ42) fibrils, leading to neuronal death in Alzheimer's disease, the possible mechanisms of amyloids degradation after exposure to factors of different nature (proteins with chaperone and protease activity, denaturant, and ultrasound) was demonstrated. Our study shows that, regardless of the method of fibril degradation, the resulting species retain some amyloid's properties, including cytotoxicity, which may even be higher than that of intact amyloids. Conclusion: The results of our work indicate that the degradation of amyloid fibrils in vivo should be treated with caution since such an approach can lead not to recovery, but to aggravation of the disease.

Microwave irradiation of corn kernels: Effects on structural, thermal, functional and rheological properties of corn flour

The current study investigated the impact of microwave treatment on the physicochemical properties of corn kernels. Three microwave powers (2, 3, 4 W/g) and three treatment times (2, 3, 4 min) were used to treat the corn kernels. The findings indicate that heat treatment increased gelatinization temperature and gelatinization enthalpy. Moreover, relative crystallinity was reduced by 17.89% in the microwave-treated flour in comparison to untreated samples, and alterations were observed in the protein secondary structure, ordered structure, and functional properties of corn flour. Microwave treatment caused the darkening of the sample and a larger particle size distribution. Higher radiation times and microwave powers exhibited more intensive treatment outcomes. Rheological tests indicated that the elastic and viscous moduli of the microwave treated corn gels were higher than those of the control group, and their large-amplitude oscillatory shear curves exhibited a strain-weak strain overshoot (type III). The results of this study suggest that microwave treatment can potentially be utilized as a technique to regulate the technical functional properties and viscoelasticity of corn flour.

Transformation and expressional studies of GaZnF gene to improve drought tolerance in Gossypium hirsutum

Drought stress is the major limiting factor in plant growth and production. Cotton is a significant crop as textile fiber and oilseed, but its production is generally affected by drought stress, mainly in dry regions. This study aimed to investigate the expression of Zinc finger transcription factor’s gene (GaZnF) to enhance the drought tolerance in Gossypium hirsutum. Sequence features of the GaZnF protein were recognized through different bioinformatics tools like multiple sequence alignment analysis, phylogenetic tree for evolutionary relationships, Protein motifs, a transmembrane domain, secondary structure and physio-chemical properties indicating that GaZnF is a stable protein. CIM-482, a local Gossypium hirsutum variety was transformed with GaZnF through Agrobacterium-mediated transformation method with 2.57% transformation efficiency. The integration of GaZnF was confirmed through Southern blot showing 531 bp, and Western blot indicated a 95 kDa transgene-GUS fusion band in transgenic plants. The normalized real-time expression analysis revealed the highest relative fold spatial expression of cDNA of GaZnF within leaf tissues at vegetative and flowering stages under drought stress. Morphological, physiological and biochemical parameters of transgenic cotton plants at 05- and 10-day drought stress was higher than those of non-transgenic control plants. The values of fresh and dry biomass, chlorophyll content, photosynthesis, transpiration rate, and stomatal conductance reduced in GaZnF transgenic cotton plants at 05- and 10-day drought stress, but their values were less low in transgenic plants than those of non-transgenic control plants. These findings showed that GaZnF gene expression in transgenic plants could be a valuable source for the development of drought-tolerant homozygous lines through breeding.

Structural and thermodynamic consequences of the G31U and U5C mutations in the SARS-CoV-2 s2m.

Within the 3’-untranslated region of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome, the stem-loop II motif (s2m) RNA element harbors two mutations (U5C and G31U) that alter the secondary structure and suspected roles in the viral life cycle compared to the SARS-CoV s2m sequence. From previously reported NMR and homodimerization experiments, the G31U mutation alone gives rise to a secondary structure and dimerization properties similar to SARS-CoV-2 s2m, while the U5C mutation was found to be more characteristic of SARS-CoV s2m. The individual effects of each mutation on the structure, dynamics, and thermodynamics have not been reported and could guide the discovery of potential functional differences of s2m in distinct viral genomes. Unbiased 3.5 microsecond molecular dynamics simulations were performed using previously reported three-dimensional atomistic coordinates based on NMR NOE assignments and the SARS-CoV s2m 1XJR crystallographic coordinates for each U5C and G31U mutation independently. Tertiary structural dependence on temperature was investigated by running simulations at 283 and 310 K to represent NMR and physiological temperatures, respectively. Principal component analysis was used to identify s2m conformational substates, and a quasiharmonic partition function was used to estimate the absolute entropy associated with each model to quantitate the structural and dynamical impact of each individual mutation. Starting with the SARS-CoV-2 s2m structure, the G31U simulation remains consistent base-pairing with our 1H NMR spectrum, while the U5C model loses the base pairing pattern. Our work addresses the structural and terminal loop entropy differences as interpretations for previously reported NMR and homodimerization experiments. Overall, a quantitative structural and dynamical insight with atomistic resolution for each U5C and G31U mutation contribution is provided for the experimentally observed deviations between SARS-CoV and SARS-CoV-2 s2m.

A crosslinked hydrogel based on recombinant human elastin for rapid repair of photodamaged skin

<p indent="0mm">Photodamage is the most common skin disease, affecting more than a third of the global population each year. Elastin in dermis plays a critical role in maintaining skin shape, and its destruction is a prominent feature in skin photodamage. Currently, elastin is mostly derived from animal tissue, which has drawbacks such as virus transmission and easy degradation. We constructed a novel Recombinant Human Elastin (RHE) and its cross-linked hydrogel to rapidly repair photodamaged skin. RHE, composed of human elastin domains, preserves a similar secondary structure and reversible phase transition properties as native. RHE was cross-linked by THPC to form hydrogels with remarkable mechanical strength. Scanning electron microscopy results showed that the RHE hydrogel has a uniformly distributed mesh structure. Cell experiments indicated that the hydrogel had no cytotoxicity and significantly promoted the proliferation of fibroblasts. Animal experiments showed that the hydrogel significantly accelerated the healing of UV-irradiated skin in mice, reducing the inflammatory response and inhibiting abnormal epidermal hyperplasia. The highly biocompatible and bioactive RHE and its cross-linked hydrogel provides an efficient treatment for photodamaged skin, which may have promising applications in dermatology and tissue engineering.

Leveraging peptide-cellulose interactions to tailor the hierarchy and mechanics of peptide-polymer hybrids.

Inspired by spider silk's hierarchical diversity, we leveraged peptide motifs with the capability to tune structural arrangement for controlling the mechanical properties of a conventional polymer framework. The addition of nanofiller with hydrogen bonding sites was used as another pathway towards hierarchical tuning via matrix-filler interactions. Specifically, peptide-polyurea hybrids (PPUs) were combined with cellulose nanocrystals (CNCs) to develop mechanically-tunable nanocomposites via tailored matrix-filler interactions (or peptide-cellulose interactions). In this material platform, we explored the effect of these matrix-filler interactions on the secondary structure, hierarchical ordering, and mechanical properties of the peptide hybrid nanocomposites. Interactions between the peptide matrix and CNCs occur in all of the PPU/CNC nanocomposites, preventing α-helical ordering, but promoting inter-molecular hydrogen bonded β-sheet formation. Depending on peptide and CNC content, the Young's modulus varies from 10 to 150 MPa. Unlike conventional cellulose-reinforced polymer nanocomposites, the mechanical properties of these composite materials are dictated by a balance of CNC reinforcement, peptidic ordering, and microphase-separated morphology. This research highlights that leveraging peptide-cellulose interactions is a strategy to create materials with targeted mechanical properties for a specific application using a limited selection of building blocks.

Exploring the Amino-Acid Composition, Secondary Structure, and Physicochemical and Functional Properties of Chickpea Protein Isolates.

This study examined the amino-acid profile, secondary structure, and physicochemical and functional properties of proteins isolated from Anatolian chickpea landraces. Secondary objective of the study was to determine whether a relationship exists between the amino-acid composition and physicochemical and functional properties. Aspartic acid and glutamic acid were the dominant amino acids, while the isolates were deficient in methionine. Secondary structures were determined by Fourier transform infrared spectroscopy, where the β-sheet was shown to be dominant. The denaturation temperature of the isolates was between 87 and 145 °C, and the highest net surface charge (≃28.6 mV) and solubility (∼95.0%) were observed at pH 9.0-10.0. The isolates' water-holding capacity varied between 2.1 and 2.7 g water/g protein, whereas their oil-holding capacity ranged between 3.4 and 4.4 g oil/g protein. Emulsion capacity, emulsifying activity, and the stability indices of isolates were found to be between 401.2 and 469.1 g oil/g protein, 14.5 and 25.7 m2/g, and 45.7 and 146.9 min, respectively. Isolates of Hisar and Erzincan chickpeas exhibited good emulsifying properties. The Yasa isolate had a relatively high hydrophobic amino-acid content and delivered the best gelation performance. Overall, significant differences in the characteristics of proteins were observed among the different chickpea landraces studied.

Conformational switch and multiple supramolecular structures of a newly identified self-assembling protein-mimetic peptide from Pseudomonas aeruginosa YeaZ protein.

Protein-mimetic peptides (PMPs) are shorter sequences of self-assembling proteins, that represent remarkable building blocks for the generation of bioinspired functional supramolecular structures with multiple applications. The identification of novel aminoacidic sequences that permit the access to valuable biocompatible materials is an attractive area of research. In this work, in silico analysis of the Pseudomonas aeruginosa YeaZ protein (PaYeaZ) led to the identification of a tetradecapeptide that represents the shortest sequence responsible for the YeaZ-YeaZ dimer formation. Based on its sequence, an innovative 20-meric peptide, called PMP-2, was designed, synthesized, and characterized in terms of secondary structure and self-assembly properties. PMP-2 conserves a helical character and self-assembles into helical nanofibers in non-polar solvents (DMSO and trifluoroethanol), as well as in dilute (0.5mM) aqueous solutions. In contrast, at higher concentrations (>2mM) in water, a conformational transition from α-helix to β-sheet occurs, which is accompanied by the Protein-mimetic peptide aggregation into 2D-sheets and formation supramolecular gel in aqueous environment. Our findings reveal a newly identified Protein-mimetic peptide that could turn as a promising candidate for future material applications.

Multi Epitopic Peptide Based Vaccine Development Targeting Immobilization Antigen of Ichthyophthirius multifiliis: A Computational Approach.

The online version contains supplementary material available at 10.1007/s10989-022-10475-1.

Overproduction and purification of Mycobacterium tuberculosis WhiB3 in Escherichia coli is enhanced by co-expression with trigger factor chaperone

Members of the WhiB-like (Wbl) family of proteins are found in Acintomycetes and are somewhat recalcitrant to overproduction as soluble proteins in the laboratory protein expression workhorse Esherichia coli. The aim of this study was to evaluate the effects of culture conditions and co-expression of the chaperone protein, trigger factor (TF), on the soluble production of recombinant Mycobacterium tuberculosis (Mtb) WhiB3. A pET28a derived expression plasmid coding for His6-WhiB3 was created and the effects of varying the concentration of inducer (IPTG), the timing of induction, the nature of the inducer (auto-induction medium) and the temperature of the cultivation on the production of soluble His6-WhiB3 were tested. Whilst His6-WhiB3 protein was readily detected, the overwhelming majority of the protein was present in the insoluble fraction of cell-free extracts. However, co-expression of the tig from pTf16, coding for TF, increased His6-WhiB3 solubility dramatically, facilitating its isolation by affinity chromatography. Purified His6-WhiB3 was shown to be monomeric, and UV–visible spectra suggested that ∼10% of the isolated protein possessed a [4Fe–4S] cluster. The secondary structural properties of His6-WhiB3 were altered by acquisition of an iron-sulfur cluster. By developing a protocol to readily overproduce and purify WhiB3, this study paves the way for future structure-function experiments.

Bioinformatic Assessment of Factors Affecting the Correlation between Protein Abundance and Elongation Efficiency in Prokaryotes.

Protein abundance is crucial for the majority of genetically regulated cell functions to act properly in prokaryotic organisms. Therefore, developing bioinformatic methods for assessing the efficiency of different stages of gene expression is of great importance for predicting the actual protein abundance. One of these steps is the evaluation of translation elongation efficiency based on mRNA sequence features, such as codon usage bias and mRNA secondary structure properties. In this study, we have evaluated correlation coefficients between experimentally measured protein abundance and predicted elongation efficiency characteristics for 26 prokaryotes, including non-model organisms, belonging to diverse taxonomic groups The algorithm for assessing elongation efficiency takes into account not only codon bias, but also number and energy of secondary structures in mRNA if those demonstrate an impact on predicted elongation efficiency of the ribosomal protein genes. The results show that, for a number of organisms, secondary structures are a better predictor of protein abundance than codon usage bias. The bioinformatic analysis has revealed several factors associated with the value of the correlation coefficient. The first factor is the elongation efficiency optimization type—the organisms whose genomes are optimized for codon usage only have significantly higher correlation coefficients. The second factor is taxonomical identity—bacteria that belong to the class Bacilli tend to have higher correlation coefficients among the analyzed set. The third is growth rate, which is shown to be higher for the organisms with higher correlation coefficients between protein abundance and predicted translation elongation efficiency. The obtained results can be useful for further improvement of methods for protein abundance prediction.

Injectable PEG-induced silk nanofiber hydrogel for vancomycin delivery

Silk fibroin nanofiber hydrogels mimicking the extracellular matrix structure have gained increased attention in the field of tissue engineering. In this study, an amorphous silk fibroin nanofiber solution was prepared using a new dissolving technique. The keys to the dissolving method include the preparation of nanofibrous silk film by dissolving silk in a CaCl2-Formic Acid (Ca-FA) solution with subsequent dissolution in a CaCl2-Ethanol-H2O ternary solution at room temperature. The resulting solution was primarily composed of native silk nano/microfibers, which primarily form amorphous structures. The amorphous silk fibroin nanofiber solution was further processed into an injectable hydrogel through a simple physical blend with polyethylene glycol (PEG). The nanofiber structure and PEG accelerated silk gelation, and the fastest gelation time was 150 s at concentrations of 5% silk and 85% PEG. The morphology, secondary structure, and rheological properties of injectable silk fibroin nanofiber hydrogels were subsequently characterized. Silk gelation was accompanied by a structural transition from random coil to β-sheet, and the resulting injectable hydrogel had an irregular lamellar structure. The antibiotic vancomycin (VANCO) was loaded in the injectable hydrogel, and the release of VANCO from the injectable hydrogel was assessed at pH values of 4.5 and 7.4. The VANCO-loaded injectable silk fibroin nanofiber hydrogel exhibited superior antibacterial properties against bacteria. A cytotoxicity test preliminarily demonstrated that the VANCO-loaded injectable silk fibroin nanofiber hydrogel had good biocompatibility for applications in tissue engineering.

Abstract LB151: Deep learning - based protein therapeutics design to treat HER2+ cancers

Abstract Background: HER2 amplification and activation occurs in approximately 15~25% of all breast cancers and is further indicated in other cancer types with high un-met medical needs, such as cholangiocarcinoma, ovarian and esophageal cancer. Further improvement of current HER2 targeting therapeutics on safety and efficacy remains to be desired. Deep learning - based computational approach, such as AlphaFold, has been successfully applied to the understanding of protein structure. However, its applications to protein therapeutic design remain largely underdeveloped. Method: We have developed an end-to-end protein design engine Apollo, which can be used to design target-binding protein therapeutics in silico. Apollo uses recurrent neural networks (RNNs), transformers and graph convolution neural networks (GCNs) to represent proteins, protein structures and protein-protein interactions. This is further improved by multi-head and hierarchical attention layers. Using generative AI, Apollo can either generate putative binding proteins de novo, or to optimize protein lead molecules. Candidate proteins are evaluated in silico for binding affinity and specificity, secondary and tertiary structure, isoelectric point, solubility, and other drug-like properties. Generated designs are iteratively improved by Apollo via a reinforcement learning loop. Results: Apollo has achieved high prediction accuracy by in silico evaluations, using metrics such as AUC and the confusion matrices. Using Apollo, we then designed mini protein binders of 50~100 aa to target HER2 extracellular domain (ECD). These binders can be produced with high yield and purity using mammalian protein expression systems. From de novo generation, the designed candidates are shown to bind to human HER2 with EC50 in the nanomolar range by ELISA. From optimizations based on lead molecules, the designed candidates have shown improved production yield and thermostability while maintaining binding affinity. Conclusions: We have shown the proof-of-concept success of using machine learning to design protein therapeutics. Citation Format: Ziwei Liang, Tom Murray, Max Bluestone, Chris Zoumadakis, Luke Martin, Alexey Vlasenko, Jeffrey LaFrence, John Lazar, Matthias Denecke, David Longo. Deep learning - based protein therapeutics design to treat HER2+ cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB151.

Rational Design of Plant Hairpin-like Peptide EcAMP1: Structural–Functional Correlations to Reveal Antibacterial and Antifungal Activity

Plant antimicrobial peptides from the α-hairpinins family (hairpin-like peptides) are known to possess a wide range of biological activities. However, less is known about the structural determinants of their antimicrobial activity. Here, we suggest that spatial structure as well as surface charge and hydrophobicity level contribute to the antimicrobial properties of α-hairpinin EcAMP1 from barnyard grass (Echinochloa cruss-galli) seeds. To examine the role of the peptide spatial structure, two truncated forms of EcAMP1 restricted by inner and outer cysteine pairs were synthesized. It was shown that both truncated forms of EcAMP1 lost their antibacterial activity. In addition, their antifungal activity became weaker. To review the contribution of surface charge and hydrophobicity, another two peptides were designed. One of them carried single amino acid substitution from tryptophan to alanine residue at the 20th position. The second one represented a truncated form of the native EcAMP1 lacking six C-terminal residues. But the α-helix was kept intact. It was shown that the antifungal activity of both modified peptides weakened. Thereby we can conclude that the secondary structural integrity, hydrophobic properties, and surface charge all play roles in the antimicrobial properties of α-hairpinins. In addition, the antibacterial activity of cereal α-hairpinins against Gram-positive bacteria was described for the first time. This study expands on the knowledge of structure–function interactions in antimicrobial α-hairpinins.

Lectin isolated from the red marine alga Solieria filiformis (Kützing) P.W. Gabrielson: Secondary structure and antidepressant-like effect in mice submitted to the lipopolysaccharide-induced inflammatory model of depression

Inflammation and oxidative and nitrosative stresses are linked to depression pathogenesis. In this context, lipopolysaccharide (LPS) has been used to induce acute inflammation and microglia activation, being considered a valuable model of depression. Therefore, the development of new medicines with greater efficiency and fewer side effects for managing depression is relevant. Among these medicines are compounds of natural origin, such as lectins. Previous studies have shown that the lectin from marine alga Solieria filiformis (SfL) has anti-inflammatory and antidepressant-like effects. Hence, it aimed to test the SfL effects against LPS-induced depressive-like behavior in mice and putative antioxidant mechanisms related to this effect and elucidate the secondary structure properties of this lectin. First, SfL was purified through sequential chromatography, and its secondary structure was analyzed by circular dichroism spectroscopy. Then, SfL (9 mg/kg; intravenously–i.v.) was administered 30 min before LPS (pre-LPS) or 1.5 and 23.5 h following LPS (post-LPS) intraperitoneal (i.p.) exposure in male Swiss mice. Saline and bupropion (30 mg/kg, i.p.) were used as controls. After the behavioral tests, mice were euthanized, and the brain areas prefrontal cortex, hippocampus, and striatum were dissected for determining oxidative and nitrosative alterations. As a result, the secondary structure of SfL is mainly composed of antiparallel β-sheets, is thermally stable up to 45 °C, and has no renaturation capacity after thermal denaturation. Furthermore, SfL prevented and reversed the behavioral and neurochemical changes LPS-induced. Thus, the present study provides a theoretical basis for an algal lectin as a promising therapeutic strategy for depression treatment.

Effect of microwave heating time on the gel properties of chicken myofibrillar proteins and their formation mechanism

SummaryThe present work was conducted to illustrate the mechanism of gel formation of myofibrillar proteins (MPs) under different microwave heating times. The results showed that the denaturation enthalpy (ΔH) of the MPs significantly decreased when the heating time increased from 3 to 9 s and then completely disappeared as the heating time progressed, indicating that the MPs gradually denatured and subsequently aggregated with increasing heating time, which was further verified by the changes in the secondary structure, electrophoretic bands, and gel properties (e.g., water holding capacity and textural profiles) of the MPs. Microstructural images indicated that the MP gel formed under 12 s had the most compact network, indicating that extended microwave heating time could induce quality deterioration of MP gels. Moreover, the hydrophobic forces, electrostatic forces, and disulphide bonds of the MPs gradually intensified with increasing microwave heating time, suggesting that both non‐covalent and covalent bonds could promote molecular denaturation and subsequent aggregation of MPs. In addition, correlation analysis revealed that the changes in the molecular conformation of MPs induced by different microwave heating times could effectively regulate the formation of MP gels and their related properties.

Reaching alignment-profile-based accuracy in predicting protein secondary and tertiary structural properties without alignment

Protein language models have emerged as an alternative to multiple sequence alignment for enriching sequence information and improving downstream prediction tasks such as biophysical, structural, and functional properties. Here we show that a method called SPOT-1D-LM combines traditional one-hot encoding with the embeddings from two different language models (ProtTrans and ESM-1b) for the input and yields a leap in accuracy over single-sequence-based techniques in predicting protein 1D secondary and tertiary structural properties, including backbone torsion angles, solvent accessibility and contact numbers for all six test sets (TEST2018, TEST2020, Neff1-2020, CASP12-FM, CASP13-FM and CASP14-FM). More significantly, it has a performance comparable to profile-based methods for those proteins with homologous sequences. For example, the accuracy for three-state secondary structure (SS3) prediction for TEST2018 and TEST2020 proteins are 86.7% and 79.8% by SPOT-1D-LM, compared to 74.3% and 73.4% by the single-sequence-based method SPOT-1D-Single and 86.2% and 80.5% by the profile-based method SPOT-1D, respectively. For proteins without homologous sequences (Neff1-2020) SS3 is 80.41% by SPOT-1D-LM which is 3.8% and 8.3% higher than SPOT-1D-Single and SPOT-1D, respectively. SPOT-1D-LM is expected to be useful for genome-wide analysis given its fast performance. Moreover, high-accuracy prediction of both secondary and tertiary structural properties such as backbone angles and solvent accessibility without sequence alignment suggests that highly accurate prediction of protein structures may be made without homologous sequences, the remaining obstacle in the post AlphaFold2 era.