Protein Structure Research Articles

GRACE: Generative Redesign in Artificial Computational Enzymology.

Designing de novo enzymes is complex and challenging, especially to maintain the activity. This research focused on motif design to identify the crucial domain in the enzyme and uncovered the protein structure by molecular docking. Therefore, we developed a Generative Redesign in Artificial Computational Enzymology (GRACE), which is an automated workflow for reformation and creation of the de novo enzymes for the first time. GRACE integrated RFdiffusion for structure generation, ProteinMPNN for sequence interpretation, CLEAN for enzyme classification, and followed by solubility analysis and molecular dynamic simulation. As a result, we selected two gene sequences associated with carbonic anhydrase from among 10,000 protein candidates. Experimental validation confirmed that these two novel enzymes, i.e., dCA12_2 and dCA23_1, exhibited favorable solubility, promising substrate-active site interactions, and achieved activity of 400 WAU/mL. This workflow has the potential to greatly streamline experimental efforts in enzyme engineering and unlock new avenues for rational protein design.

Assessing the Utility of ColabFold and AlphaMissense in Determining Missense Variant Pathogenicity for Congenital Myasthenic Syndromes

Background/Objectives: Congenital myasthenic syndromes (CMSs) are caused by variants in >30 genes with increasing numbers of variants of unknown significance (VUS) discovered by next-generation sequencing. Establishing VUS pathogenicity requires in vitro studies that slow diagnosis and treatment initiation. The recently developed protein structure prediction software AlphaFold2/ColabFold has revolutionized structural biology; such predictions have also been leveraged in AlphaMissense, which predicts ClinVar variant pathogenicity with 90% accuracy. Few reports, however, have tested these tools on rigorously characterized clinical data. We therefore assessed ColabFold and AlphaMissense as diagnostic aids for CMSs, using variants of the CHRN genes that encode the nicotinic acetylcholine receptor (nAChR). Methods: Utilizing a dataset of 61 clinically validated CHRN variants, (1) we evaluated the possibility of a ColabFold metric (either predicted structural disruption, prediction confidence, or prediction quality) that distinguishes variant pathogenicity; (2) we assessed AlphaMissense’s ability to differentiate variant pathogenicity; and (3) we compared AlphaMissense to the existing pathogenicity prediction programs AlamutVP and EVE. Results: Analyzing the variant effects on ColabFold CHRN structure prediction, prediction confidence, and prediction quality did not yield any reliable pathogenicity indicative metric. However, AlphaMissense predicted variant pathogenicity with 63.93% accuracy in our dataset—a much greater proportion than AlamutVP (27.87%) and EVE (28.33%). Conclusions: Emerging in silico tools can revolutionize genetic disease diagnosis—however, improvement, refinement, and clinical validation are imperative prior to practical acquisition.

The Knob Domain of the Fiber-1 Protein Affects the Replication of Fowl Adenovirus Serotype 4

Fowl adenovirus serotype 4 (FAdV-4) outbreaks have caused significant economic losses in the Chinese poultry industry since 2015. The relationships among viral structural proteins in infected hosts are relatively unknown. To explore the role of different parts of the fiber-1 protein in FAdV-4-infected hosts, we truncated fiber-1 into fiber-1-Δ1 (73–205 aa) and fiber-1-Δ2 (211–412 aa), constructed pEF1α-HA-fiber-1-Δ1 and pEF1α-HA-fiber-1-Δ2 and then transfected them into leghorn male hepatocyte (LMH) cells. After FAdV-4 infection, the roles of fiber-1-Δ1 and fiber-1-Δ2 in the replication of FAdV-4 were investigated, and transcriptome sequencing was performed. The results showed that the fiber-1-Δ1 and fiber-1-Δ2 proteins were the shaft and knob domains, respectively, of fiber-1, with molecular weights of 21.4 kDa and 29.6 kDa, respectively. The fiber-1-Δ1 and fiber-1-Δ2 proteins were mainly localized in the cytoplasm of LMH cells. Fiber-1-Δ2 has a greater ability to inhibit FAdV-4 replication than fiber-1-Δ1, and 933 differentially expressed genes (DEGs) were detected between the fiber-1-Δ1 and fiber-1-Δ2 groups. Functional analysis revealed these DEGs in a variety of biological functions and pathways, such as the phosphoinositide 3-kinase–protein kinase b (PI3K–Akt) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, cytokine–cytokine receptor interactions, Toll-like receptors (TLRs), the Janus tyrosine kinase–signal transducer and activator of transcription (Jak–STAT) signaling pathway, the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) signaling pathway, and other innate immune pathways. The mRNA expression levels of type I interferons (IFN-α and INF-β) and proinflammatory cytokines (IL-1β, IL-6 and IL-8) were significantly increased in cells overexpressing the fiber-1-Δ2 protein. These results demonstrate the role of the knob domain of the fiber-1 (fiber-1-Δ2) protein in FAdV-4 infection and provide a theoretical basis for analyzing the function of the fiber-1 protein of FAdV-4.

Insight into the conformational changes and allergenic reactivity of coarse wheat bran induced by ozone treatment.

Whole grain food has gradually come into view because of its high nutritional value, but the incidence of wheat allergies has been increasing year by year. Ozone is a new non-thermal desensitization technology in food processing, and its effect on coarse wheat bran protein has received little study. We investigated the effects of different ozone airflow rates and treatment times on wheat bran allergenic property and protein structure. The ozone treatment time of 30 min at an airflow rate of 5 L min-1 reduced the coarse wheat bran allergenic property by 61.14%; subunits of 33, 46, 48 and 68 kDa were significantly less allergenic via western blotting; and the lowest releasing rate of β-hexosaminidase was 25.32% in the cell model of rat basophilic leukaemia 2H3. According to secondary structure and chemical interaction determination, the protein molecules were reorganized and aggregated by disulfide bonds and hydrophobic contacts following ozone exposure. Ozone treatment has a beneficial potential in reducing the allergenic property of coarse wheat bran and improving the safety of whole wheat products. © 2024 Society of Chemical Industry.

Stable and Minimum Size Solubilization of Membrane Proteins with Cocktails of Phospholipid Analogues.

Membrane proteins (MPs) play important roles in various cellular processes and are major targets for drugs. Solubilization of MPs is often needed for structural and biophysical studies. For high-resolution nuclear magnetic resonance measurements, there is a size limit of the sample (<100 kDa), and a high thermal stability at an increased temperature is required. Furthermore, lipid bilayer-like environments are desirable to preserve the native states of MPs. However, existing solubilization techniques do not fulfill these requirements at the same time. In this study, we combined two phospholipid analogues as a solubilizer and stabilizer to isolate MPs. This method maintained bacteriorhodopsin (bR) extracted from purple membranes in its native state for 7 d at 40 °C. The solubility was comparable to that of conventional detergents for MPs, and the thermal stability of the solubilizate was the best among them. The increase in the molecular size caused by the solubilization of bR was only 20 kDa, indicating that 20 phospholipid analogue molecules were sufficient to solubilize one bR molecule. 15N-1H heteronuclear single quantum coherence spectra of solubilized 2H- and 15N-labeled bR gave ∼80% of the expected peaks. In addition, the lysate of human neuropeptide Y2 receptor-expressing mammalian cells exhibited ligand recognition for 7 d at 37 °C, suggesting that this technique can be used for ligand screening. Moreover, the structure of the single membrane-spanning M2 protein of the influenza A virus expressed in Escherichia coli was stably maintained for 7 d at 40 °C. Thus, our method is promising for various MP studies.

Crystallographic insights into lipid-membrane protein interactions in microbial rhodopsins

The primary goal of our work is to provide structural insights into the influence of the hydrophobic lipid environment on the membrane proteins (MPs) structure and function. Our work will not cover the well-studied hydrophobic mismatch between the lipid bilayer and MPs. Instead, we will focus on the less-studied direct molecular interactions of lipids with the hydrophobic surfaces of MPs. To visualize the first layer of amphiphiles surrounding MPs and analyze their interaction with the proteins, we use the available highest-quality crystallographic structures of microbial rhodopsins. The results of the structure-based analysis allowed us to formulate the hypothetical concept of the role of the nearest layer of the lipids as an integral part of the MPs that are important for their structure and function. We then discuss how the lipid-MPs interaction is influenced by exogenous hydrophobic molecules, noble gases, which can compete with lipids for the surface of MPs and can be used in the systematic approach to verify the proposed concept experimentally. Finally, we raise the problems of currently available structural data that should be overcome to obtain a more profound picture of the lipid-MP interactions.

Noncanonical Amino Acid Tools and Their Application to Membrane Protein Studies.

Methods rooted in chemical biology have contributed significantly to studies of integral membrane proteins. One recent key approach has been the application of genetic code expansion (GCE), which enables the site-specific incorporation of noncanonical amino acids (ncAAs) with defined chemical properties into proteins. Efficient GCE is challenging, especially for membrane proteins, which have specialized biogenesis and cell trafficking machinery and tend to be expressed at low levels in cell membranes. Many eukaryotic membrane proteins cannot be expressed functionally in E. coli and are most effectively studied in mammalian cell culture systems. Recent advances have facilitated broader applications of GCE for studies of membrane proteins. First, AARS/tRNA pairs have been engineered to function efficiently in mammalian cells. Second, bioorthogonal chemical reactions, including cell-friendly copper-free "click" chemistry, have enabled linkage of small-molecule probes such as fluorophores to membrane proteins in live cells. Finally, in concert with advances in GCE methodology, the variety of available ncAAs has increased dramatically, thus enabling the investigation of protein structure and dynamics by multidisciplinary biochemical and biophysical approaches. These developments are reviewed in the historical framework of the development of GCE technology with a focus on applications to studies of membrane proteins.

Solvation phenomena in ternary system tetramethylurea-methylacetamide-water: Insights from volumetric, compressibility and FTIR analysis

The properties of the ternary systems N,N,N',N'-tetramethylurea - N-methylacetamide - water were investigated using Fourier-transform infrared spectroscopy (FTIR), volumetric and compression measurements. Densities and sound velocities were determined in order to obtain the apparent molar volumes (VΦ) and apparent molar isentropic compressions (▪S,Φ). These values were then extrapolated to infinite dilution. Additionally, interaction parameters were calculated from the McMillan-Mayer theory. The studies were conducted at 288.15, 298.15, and 308.15 K, at atmospheric pressure (0.1 MPa). The concentration ranges of N-methylacetamide were 2, 4, 6, and 8 moles per kilogram of pure water, and for N,N,N',N'-tetramethylurea from 0 to around 0.35 moles per kilogram of solvent. Discrete changes in isentropic compression were observed. This is the result of the alignment of plots of ▪S,Φ0 as a function of NMA concentration. The results for N,N,N',N'-tetramethylurea were compared with analogous data for the system containing n-butylurea, which is an isomeric compound but exhibits different hydration behaviour. Additionally, large volumetric interaction coefficients were observed, indicating strong interactions between urea derivatives and NMA. This suggests the possibility of strong interactions between protein destabilizers and the protein backbone, differing from those observed for protein structure stabilizers. The observation contributes to understanding the mechanism of osmolyte action and their influence on protein stability.

Critical Assessment of Protein Engineering (CAPE): A Student Challenge on the Cloud.

The success of AlphaFold in protein structure prediction highlights the power of data-driven approaches in scientific research. However, developing machine learning models to design and engineer proteins with desirable functions is hampered by limited access to high-quality data sets and experimental feedback. The Critical Assessment of Protein Engineering (CAPE) challenge addresses these issues through a student-focused competition, utilizing cloud computing and biofoundries to lower barriers to entry. CAPE serves as an open platform for community learning, where mutant data sets and design algorithms from past contestants help improve overall performance in subsequent rounds. Through two competition rounds, student participants collectively designed >1500 new mutant sequences, with the best-performing variants exhibiting catalytic activity up to 5-fold higher than the wild-type parent. We envision CAPE as a collaborative platform to engage young researchers and promote computational protein engineering.

Early Enteral Feeding Restores Neurofilament Light Chain Serum Levels in Preterm Newborns.

Positive effects of early nutritional strategies on neurological outcomes have been observed when nutrients were administered by the enteral route, especially during the first week of life. Evidence reports that serum neurofilament light chain (NfL), a structural protein of neurons, is a specific and reliable biomarker of neuronal damage. The present study aimed to investigate the effect of early enteral nutrition (EN) in minimizing neuroaxonal damage and assessing NfL serum levels in preterm neonates. Fifty-four preterm neonates without severe brain impairment and 20 full-term babies as controls were enrolled from the Neonatal Intensive Care Unit at the Policlinico Umberto I in Rome. We performed blood sampling at birth (day of life 0 - DoL 0) in 20 full-term newborns and in 19 pre-term infants. Furthermore, we executed blood sampling at DoL 28 in other 22 pre-term newborns who received early enteral nutrition (EN) within the third DoL (Early-EN) and in 13 other pre-term newborns who received EN after the third DoL (Late-EN). Serum levels of NfL were higher in preterm babies when compared to full-term neonates, at DoL 0 (48.81 ± 9.4 vs. 11.67 ± 1.4 pg/ml; p = 0.007). Interestingly, at DoL 28, serum NfL was significantly decreased in the Early-EN newborns compared to the Late-EN groups (15.22 ± 2.0 vs. 50.05 ± 17.9 pg/ml; p = 0.03). It was shown that early enteral feeding, within the first week of life, could be a useful tool for limiting neurological impairment in pre-term neonates by restoring NfL.

Conformational and processing properties of a high-active ingredient involving soy protein isolates bound with anthocyanins and its application in cake baking

Soybean protein is of plant origin and is commonly appropriate for improving the processing characteristics of foods. This study aimed to explore a novel functional ingredient that contained soybean protein isolate (SPI) and blueberry anthocyanins (BANs). The spatial conformation and secondary structure of SPI-BANs complexes were analyzed using circular dichroism and fluorescence spectroscopy, the processing properties were investigated as well as the retention of antioxidant activity during thermal treatments. Results showed that the contents of free sulfhydryl and free amino groups in complexes increased to 3.50 and 1.19 folds than those of SPI, respectively, while the surface hydrophobicity decreased by 74.23%. Compared with SPI, the BANs-modified SPI had a smaller particle size of 29.12 nm and a lower zeta-potential of -8.73 mV and on the other hand, the complexes possessed higher solubility (83.08%) and foaming and emulsifying properties (115.08% and 54.03 m2/g). After fortification with SPI-BANs, the baking loss rate and adhesiveness of chiffon cake were reduced by 10.82% and improved to 0.24 N.mm, respectively. The high antioxidant activities of SPI-BANs under heat led to the cake’s bioactivities largely enhanced by 1.99 ~ 12.71 folds, being 345.19 µg Trolox/g for the DPPH radical scavenging activity. This study developed the functional food ingredients as antioxidants and a substitute for animal-based proteins in bakery products, which was safe and sustainable by using the dietary components from plant resources.Graphical

Influence of (ultra-)processing methods on aquatic proteins and product quality.

Aquatic products are a high-quality source of protein for humans, and the changes in protein during aquatic product processing are crucial for nutritional value, product performance, and consumer health. With the advancement of science and technology, aquatic product processing methods have become increasingly diverse. In addition to traditional methods such as thermal processing (steaming, roasting, and frying) and pickling, emerging non-thermal processing technologies, such as high pressure, ultrasound, and irradiation, are also being applied. During (ultra-)processing, aquatic products undergo complex biochemical reactions, among which protein oxidation significantly affects the quality of aquatic products. Protein oxidation can alter the molecular structure of proteins, thereby changing their functional properties and ultimately impacting product quality. This paper primarily explored the effects of protein changes under different processing methods on aquatic product quality and human health, as well as techniques for controlling protein oxidation. It aims to provide a theoretical basis for selecting appropriate processing methods, improving aquatic product quality, and controlling protein oxidation in aquatic products, and to offer scientific guidance for practical production.

SEC-SAXS/MC Ensemble Structural Studies of the Microtubule Binding Protein Cdt1 Show Monomeric, Folded-Over Conformations.

Cdt1 is a mixed folded protein critical for DNA replication licensing and it also has a "moonlighting" role at the kinetochore via direct binding to microtubules and the Ndc80 complex. However, it is unknown how the structure and conformations of Cdt1 could allow it to participate in these multiple, unique sets of protein complexes. While robust methods exist to study entirely folded or unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. In this work, we employ orthogonal biophysical and computational techniques to provide structural characterization of mitosis-competent human Cdt1. Thermal stability analyses shows that both folded winged helix domains1 are unstable. CD and NMR show that the N-terminal and linker regions are intrinsically disordered. DLS shows that Cdt1 is monomeric and polydisperse, while SEC-MALS confirms that it is monomeric at high concentrations, but without any apparent inter-molecular self-association. SEC-SAXS enabled computational modeling of the protein structures. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble of structures. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with SAXS. The best-fit models have the N-terminal and linker disordered regions extended into the solution and the two folded domains close to each other in apparent "folded over" conformations. We hypothesize the best-fit Cdt1 conformations could be consistent with a function as a scaffold protein that may be sterically blocked without binding partners. Our study also provides a template for combining experimental and computational techniques to study mixed-folded proteins.

RNA editing-induced structural and functional adaptations of NAD9 in Triticum aestivum under drought stress

IntroductionMitochondria are essential organelles in eukaryotic cells, producing ATP through the electron transport chain to supply energy for cellular activities. Beyond energy production, mitochondria play crucial roles in cellular signaling, stress responses, and the regulation of reactive oxygen species. In plants, mitochondria are one of the keys to responding to environmental stresses which can significantly affect crop productivity, particularly in crops like wheat. RNA editing, a post-transcriptional RNA modification process in mitochondria, is linked to regulating these stress responses.MethodsThis study explores RNA editing patterns in the nad9 gene of wheat drought-tolerant (Giza168) and drought-sensitive (Gemmiza10) wheat cultivars under drought stress to understand plant adaptation mechanisms. RNA-seq data for these cultivars were analyzed using CLC Genomic Workbench to identify RNA editing sites in the nad9 gene, examining subsequent amino acid changes and predicting secondary structure modifications. These RNA editing sites were validated using qRT-PCR on drought-treated seedlings at 0, 2, and 12 hours post-treatment. Protein models were generated using AlphaFold, with functional predictions and structure verification conducted using various bioinformatics tools to investigate the effect of RNA editing on protein level.ResultsThe results showed significant RNA editing events, especially C-to-T conversions, in the nad9 gene across different drought exposure times. Giza168 had 22 editing sites, while Gemmiza10 had 19, with several showing significant differences between control and stress conditions. RNA editing influenced the NAD9 protein's secondary structure, particularly beta sheets, and 3D modeling highlighted the structural impacts of these edits. The N-terminal region of NAD9 contained important regulatory motifs, suggesting a complex regulatory environment.DiscussionThis study reveals key editing sites that differ between drought-tolerant and sensitive wheat cultivars, impacting NAD9 protein structures and highlighting the role of RNA editing in enhancing drought resilience. Additionally, the study suggests potential regulatory mechanisms, including phosphorylation and ubiquitination that influence mitochondrial stability and function.

Identification of a novel mycovirus belonging to the “flexivirus”-related family with icosahedral virion

Abstract The order Tymovirales currently comprises five viral families with positive-sense RNA ((+)RNA) genomes that infect plants, fungi, and insects. Virion morphologies within the order Tymovirales differ between families, with icosahedral virions in the Tymoviridae and filamentous virions in the other “flexi”viridae families. Despite their different morphologies, these viruses are placed in the same order based on phylogenetic analyses of replicase-associated polyproteins. However, one of the families in the Tymovirales, Deltaflexiviridae, is considered to be capsidless because there have been no published reports of virion isolation. Here we report that a new “flexivirus”-related (+)RNA virus, prospectively named Fusarium oxysporum icosahedral virus 1 (FoIV1), is icosahedral, and that most deltaflexiviruses may have icosahedral virions. Phylogenetic analyses based on replicase-associated polyproteins indicated that FoIV1 forms a distinct group in the Tymovirales with some viruses originally assigned to the Deltaflexiviridae. Electron microscopy, protein analysis, and protein structure predictions indicate that FoIV1 ORF4 encodes a single jelly-roll (SJR)-like coat protein (CP) that constitutes the icosahedral virions. Results of clustering analyses based on amino acid sequences and predicted CP structures suggested that most of the deltaflexiviruses have icosahedral virions composed of SJR-like CPs as in FoIV1, rather than having filamentous virions or capsidless. These results challenge the conventional understanding of viruses in the order Tymovirales, with important implications for revising its taxonomic framework and providing insights into the evolutionary relationships within this diverse and broad host range group of (+)RNA viruses.

Genetic diversity and spread of recombinant coxsackievirus A4 in hand, foot, and mouth disease cases in Bangkok, Thailand: 2017–2023

Coxsackievirus A4 (CVA4) has recently become one of the most common causative agents of hand, foot, and mouth disease. The current study investigated the genetic diversity and spread of recombinant CVA4 by analyzing circulating genotypes and recombinant strains in Bangkok, Thailand, from 2017 to 2023. Partial VP1, 3Dpol, and whole genome sequencing of CVA4 samples collected from collaborating hospitals were conducted. Phylogenetic analysis of CVA4 VP1 and 3Dpol genome regions revealed discordance, indicating recombination. The predominant CVA4 genotype was C3, primarily observed in 2019. The predominant genotype in 2017 was C1. D2, commonly found in China, was occasionally observed. In nucleotide similarity analysis, intertypic recombination between CVA4 and EV-A during the evolutionary history of the virus was evident, particularly in the nonstructural region. The estimated emergence of genotypes C1 and C3 in Thailand occurred around 2014, with an evolutionary rate of 5.8 × 10− 3 nucleotide substitutions per site per year. Genotype D2 exhibited notable variability across both the entire genome and the structural protein region compared to genotype C. Monitoring the genetic diversity and circulation of recombinant CVA4 is crucial for identifying newly emerging virus strains, enabling prompt public health responses and containment efforts, and enhancing surveillance in Thailand.

Structure-aware annotation of leucine-rich repeat domains.

Protein domain annotation is typically done by predictive models such as HMMs trained on sequence motifs. However, sequence-based annotation methods are prone to error, particularly in calling domain boundaries and motifs within them. These methods are limited by a lack of structural information accessible to the model. With the advent of deep learning-based protein structure prediction, existing sequenced-based domain annotation methods can be improved by taking into account the geometry of protein structures. We develop dimensionality reduction methods to annotate repeat units of the Leucine Rich Repeat solenoid domain. The methods are able to correct mistakes made by existing machine learning-based annotation tools and enable the automated detection of hairpin loops and structural anomalies in the solenoid. The methods are applied to 127 predicted structures of LRR-containing intracellular innate immune proteins in the model plant Arabidopsis thaliana and validated against a benchmark dataset of 172 manually-annotated LRR domains.

SOP-MULTI: A Self-Organized Polymer-Based Coarse-Grained Model for Multidomain and Intrinsically Disordered Proteins with Conformation Ensemble Consistent with Experimental Scattering Data.

Multidomain proteins with long flexible linkers and full-length intrinsically disordered proteins (IDPs) are best defined as an ensemble of conformations rather than a single structure. Determining high-resolution ensemble structures of such proteins poses various challenges by using tools from experimental structural biophysics. Integrative approaches combining available low-resolution ensemble-averaged experimental data and in silico biomolecular reconstructions are now often used for the purpose. However, extensive Boltzmann weighted conformation sampling for large proteins, especially for ones where both the folded and disordered domains exist in the same polypeptide chain, remains a challenge. In this work, we present a 2-site per amino-acid resolution SOP-MULTI force field for simulating coarse-grained models of multidomain proteins. SOP-MULTI combines two well-established self-organized polymer models─: (i) SOP-SC models for folded systems and (ii) SOP-IDP for IDPs. For the SOP-MULTI, we introduce cross-interaction terms between the beads belonging to the folded and disordered regions to generate conformation ensembles for full-length multidomain proteins such as hnRNP A1, TDP-43, G3BP1, hGHR-ECD, TIA1, HIV-1 Gag, polyubiquitin, and FUS. When back-mapped to all-atom resolution, SOP-MULTI trajectories faithfully recapitulate the scattering data over the range of the reciprocal space. We also show that individual folded domains preserve native contacts with respect to solved folded structures, and root-mean-square fluctuations of residues in folded domains match those obtained from all-atom molecular dynamics simulation trajectories of the same folded systems. SOP-MULTI force field is made available as a LAMMPS-compatible user package along with setup codes for generating the required files for any full-length protein with folded and disordered regions.

Influence of protein in low paste viscosities of Bambara groundnut flours from heat-treated Bambara groundnut seeds

Heat treatment of Bambara groundnut seeds has been reported to cause low paste viscosities in resulting flours. Structural changes in Bambara groundnut protein, due to heat treatment, causes the protein to encapsulate starch, making it unavailable to paste causing low paste viscosities. In this study, trypsin was used to hydrolyze proteins in the flour and the microstructure analysis confirmed the disappearance of aggregates. Flour microstructure analysis confirmed hydrolysis of protein from previously aggregated status and showed liberated individual starch granules. Following the treatment of flours by trypsin, Confocal laser scanning microscopy did not show a protein signal. Hydrolysing Bambara groundnut proteins significantly increased the flour paste viscosities (P < 0.05). The final viscosities for flours from 20 % moisture-conditioned and infrared heat-treated seeds for 5 min were 733.9 mPa s before protein hydrolysis and 2081.71 mPa s after protein hydrolysis. The gelatinization temperature (81 °C) did not show a significant change following protein hydrolysis. Sodium dodecyl-sulphate polyacrylamide gel electrophoresis band intensity increased indicative of disulfide bonding and protein polymerisation when microwave and infrared heat treatment were combined. There were changes in Bambara groundnut protein secondary structures such as an increase of 57 % in β-sheet along with a 60 % reduction in the α-helix as shown by the Fourier-transform infrared spectroscopy. The changes in secondary structure of Bambara groundnut protein were caused by microwave and infrared heating. Heat treatment of Bambara groundnut seeds is partly responsible for the reduction in paste viscosities of their flours.

Molecular dynamics simulations provide insights into ULK-101 potency and selectivity toward autophagic kinases ULK1/2

Kinase domains are highly conserved within proteins in both sequence and structure. Many factors, including phosphorylation, amino acid substitutions or mutations, and small molecule inhibitor binding, influence conformations of the kinase domain and enzymatic activity. ULK1 and ULK2 are serine/threonine kinases that serve important roles in autophagy, an intracellular recycling process capable of degrading proteins and organelles via fusion with lysosomes. ULK1/2 are emerging as therapeutic targets in human cancer, particularly KRAS-driven malignancies. Here, we performed molecular dynamics (MD) simulations to hypothesize bound poses for the ULK1/2 small molecule inhibitor, ULK-101. We observed stable bound states for ULK-101 to the adenosine triphosphate (ATP)-binding site of ULK2, coordinated by hydrogen bonding with the hinge backbone and the catalytic lysine sidechain. Notably, ULK-101 occupies a hydrophobic pocket associated with the N-terminus of the αC-helix. Large movements in the phosphate-binding loop (P-loop) are also associated with ULK-101 inhibitor binding and exit from ULK2. Together, our data support a model to explain ULK-101 potency toward ULK1/2.