Changes In Amino Acid Composition Research Articles

The Rapid Evolution of De Novo Proteins in Structure and Complex.

Recent studies in the rice genome-wide have established that de novo genes, evolving from non-coding sequences, enhance protein diversity through a stepwise process. However, the pattern and rate of their evolution in protein structure over time remain unclear. Here, we addressed these issues within a surprisingly short evolutionary timescale (<1 million years for 97% of Oryza de novo genes) with comparative approaches to gene duplicates. We found that de novo genes evolve faster than gene duplicates in the intrinsic disordered regions (IDRs, such as random coils), secondary structure elements (such as α-helix and β-strand), hydrophobicity, and molecular recognition features (MoRFs). In de novo proteins, specifically, we observed an 8-14% decay in random coils and IDR lengths and a 2.3-6.5% increase in structured elements, hydrophobicity, and MoRFs, per million years on average. These patterns of structural evolution align with changes in amino acid composition over time as well. We also revealed higher positive charges but smaller molecular weights for de novo proteins than duplicates. Tertiary structure predictions showed that most de novo proteins, though not typically well-folded on their own, readily form low-energy and compact complexes with other proteins facilitated by extensive residue contacts and conformational flexibility, suggesting a faster-binding scenario in de novo proteins to promote interaction. These analyses illuminate a rapid evolution of protein structure in de novo genes in rice genomes, originating from non-coding sequences, highlighting their quick transformation into active, protein complex-forming components within a remarkably short evolutionary timeframe.

Weak purifying selection in allelic diversity of the ADSL gene in indigenous and local chicken breeds and red junglefowl in Thailand

High levels of purine and uric acid, which are associated with health issues such as gout and cardiovascular disease, are found in the meat of fast-growing broiler chickens, which raises concerns about the quality of chicken meat and the health of the consumers who consume it. High genetic homogeneity and uniformity, particularly in genes involved in the synthesis of inosine monophosphate (IMP) and subsequent process of purine synthesis, which are associated with the meat quality, are exhibited in commercial broiler chickens owing to intensive inbreeding programs. Adenosine succinate lyase (ADSL) is a key enzyme involved in de novo purine biosynthetic pathway and its genetic polymorphisms affect IMP metabolism and purine content. In this study, we investigated the polymorphism of the ADSL gene in indigenous and local chicken breeds and red junglefowl in Thailand, using metabarcoding and genetic diversity analyses. Five alleles with 73 single nucleotide polymorphisms in exon 2, including missense and silent mutations, which may act on the synthesis efficiency of IMP and purine. Their protein structures revealed changes in amino acid composition that may affect ADSL enzyme activity. Weak purifying selection in these ADSL alleles was observed in the chicken population studied, implying that the variants have minor fitness impacts and a greater probability of fixation of beneficial mutations than strong purifying selection. A potential selective sweep was observed in Mae Hong Son chickens, whose purine content was lower than that in other breeds. This suggests a potential correlation between variations of the ADSL gene and reduced purine content and an impact of ADSL expression on the quality of chicken meat. However, further studies are required to validate its potential availability as a genetic marker for selecting useful traits that are beneficial to human health and well-being.

Omics data analysis reveals the system-level constraint on cellular amino acid composition

Proteins play a pivotal role in coordinating the functions of organisms, essentially governing their traits, as the dynamic arrangement of diverse amino acids leads to a multitude of folded configurations within peptide chains. Despite dynamic changes in amino acid composition of an individual protein (referred to as AAP) and great variance in protein expression levels under different conditions, our study, utilizing transcriptomics data from four model organisms uncovers surprising stability in the overall amino acid composition of the total cellular proteins (referred to as AACell). Although this value may vary between different species, we observed no significant differences among distinct strains of the same species. This indicates that organisms enforce system-level constraints to maintain a consistent AACell, even amid fluctuations in AAP and protein expression. Further exploration of this phenomenon promises insights into the intricate mechanisms orchestrating cellular protein expression and adaptation to varying environmental challenges.

Gamma radiation vs high pressure pretreatment on physicochemical characteristics of rice bran hydrolysate

This study investigated the effect of using gamma radiation and high-pressure processing as pretreatment, to consider the structural and amino acid composition changes in rice bran hydrolysate (RBH). The extraction yield and degree of hydrolysis of the irradiated sample were greater than those of the pressurized and control samples, which radiation at 10 kGy gave 31 % yield. Protein content of the control was the highest at 36.1 %, with 32.4 % in pressurized sample at 500 MPa. Control had the highest concentration of total and branched-chain amino acids, with a value of 25,834 mg/100g. Before and after extraction, the microstructure changed visibly and protein agglomeration can be significantly induced by applying a high-pressure. Therefore, this study showed the potential of using both pretreatment methods prior to enzymolysis extraction, with radiation producing more extract. High-pressure produced more protein content, but neither method produced any difference in amino acid content.

Polystyrene nanoplastics exposure alters muscle amino acid composition and nutritional quality of Pacific whiteleg shrimp (Litopenaeus vannamei)

Litopenaeus vannamei were exposed to 80-nm polystyrene nanoplastics (NPs) at different concentrations (0, 0.1, 1, 5, and 10 mg/L) for 28 days to study the effects on muscle nutritional quality. Our results showed that with increasing NPs concentrations, the survival rate, specific gain rate, and protein efficiency ratio decreased but the feed conversion ratio increased. There was no significant difference in moisture, ash, and crude lipid content in the muscle, and a general decrease in crude protein content was observed. However, the total amino acid and semi-essential amino acid contents decreased. The spacing between muscle fibers and the melting morphology of muscle increased. The hardness of muscle flesh texture increased, but springiness, cohesiveness, and chewiness decreased. Regarding antioxidant enzyme activity, the activity of catalase decreased, but the total antioxidant capacity, superoxide dismutase activity, and reduced glutathione first increased and then decreased. The expression level of the growth-related genes retinoid X receptor (RXR), chitin synthase (CHS), and calmodulin A (CaM) first increased then decreased, but calcium/calmodulin-dependent protein kinase I (CaMKI), ecdysteroid receptor (EcR), chitinase 5 (CHT5), cell division cycle 2 (Cdc2), and cyclin-dependent kinase 2 (CDK2) decreased. Our results suggest that exposure to NPs can inhibit growth by inducing oxidative stress, which leads to muscle tissue damage and changes in amino acid composition. These results will provide a theoretical reference for the risk assessment of NPs and the ecological health aquaculture of shrimp.

Metallic Nanocarriers for Therapeutic Peptides: Emerging Solutions Addressing the Delivery Challenges in Brain Ailments.

Peptides and proteins have recently emerged as efficient therapeutic alternatives to conventional therapies. Although they emerged a few decades back, extensive exploration of various ailments or disorders began recently. The drawbacks of current chemotherapies and irradiation treatments, such as drug resistance and damage to healthy tissues, have enabled the rise of peptides in the quest for better prospects. The chemical tunability and smaller size make them easy to design selectively for target tissues. Other remarkable properties include antifungal, antiviral, anti-inflammatory, protection from hemorrhage stroke, and as therapeutic agents for gastric disorders and Alzheimer and Parkinson diseases. Despite these unmatched properties, their practical applicability is often hindered due to their weak susceptibility to enzymatic digestion, serum degradation, liver metabolism, kidney clearance, and immunogenic reactions. Several methods are adapted to increase the half-life of peptides, such as chemical modifications, fusing with Fc fragment, change in amino acid composition, and carrier-based delivery. Among these, nanocarrier-mediated encapsulation not only increases the half-life of the peptides in vivo but also aids in the targeted delivery. Despite its structural complexity, they also efficiently deliver therapeutic molecules across the blood-brain barrier. Here, in this review, we tried to emphasize the possible potentiality of metallic nanoparticles to be used as an efficient peptide delivery system against brain tumors and neurodegenerative disorders. SIGNIFICANCE STATEMENT: In this review, we have emphasized the various therapeutic applications of peptides/proteins, including antimicrobial, anticancer, anti-inflammatory, and neurodegenerative diseases. We also focused on these peptides' challenges under physiological conditions after administration. We highlighted the importance and potentiality of metallic nanocarriers in the ability to cross the blood-brain barrier, increasing the stability and half-life of peptides, their efficiency in targeting the delivery, and their diagnostic applications.

C-terminal sequence stability profiling in Saccharomyces cerevisiae reveals protective protein quality control pathways

Protein quality control (PQC) mechanisms are essential for degradation of misfolded or dysfunctional proteins. An essential part of protein homeostasis is recognition of defective proteins by PQC components and their elimination by the ubiquitin-proteasome system, often concentrating on protein termini as indicators of protein integrity. Changes in amino acid composition of C-terminal ends arise through protein disintegration, alternative splicing, or during the translation step of protein synthesis from premature termination or translational stop-codon read-through. We characterized reporter protein stability using light-controlled exposure of the random C-terminal peptide collection (CtPC) in budding yeast revealing stabilizing and destabilizing features of amino acids at positions-5 to-1 of the C terminus. The (de)stabilization properties of CtPC-degrons depend on amino acid identity, position, as well as composition of the C-terminal sequence and are transferable. Evolutionary pressure toward stable proteins in yeast is evidenced by amino acid residues under-represented in cytosolic and nuclear proteins at corresponding C-terminal positions, but over-represented in unstable CtPC-degrons, and vice versa. Furthermore, analysis of translational stop-codon read-through peptides suggested that such extended proteins have destabilizing C termini. PQC pathways targeting CtPC-degrons involved the ubiquitin-protein ligase Doa10 and the cullin-RING E3 ligase SCFDas1 (Skp1-Cullin-F-box protein). Overall, our data suggest a proteome protection mechanism that targets proteins with unnatural C termini by recognizing a surprisingly large number of C-terminal sequence variants.

Bunch microclimate influence amino acids and phenolic profiles of Pinot noir grape berries.

The increase of temperature due to climate change at different phenological stages of grapevine has already been demonstrated to affect accumulation of primary and secondary metabolites in grape berries. This has a significant implication for Pinot noir especially in New Zealand context as these compounds can have direct and indirect effects on wine quality. This study investigates how varying bunch microclimate through changes in temperature applied at veraison stage can affect: fresh weight, total soluble solids, the accumulation of anthocyanins, total phenolics and amino acids of the grape berries. This was studied over two growing seasons (2018/19 and 2019/20) with Pinot noir vines being grown at two different temperatures in controlled environment (CE) chambers. The vines were exposed to 800 µmol/m2/s irradiance with diurnal changes in day (22°C or 30°C) and night (15°C) temperatures. This experimental set up enabled us to determine the accumulation of these metabolite at harvest (both seasons) and throughout berry development (second season). The results showed that berry weight was not influenced by temperature increase. The total soluble solids (TSS) were significantly increased at 30°C, however, this was not at the expense of berry weight (i.e., water loss). Anthocyanin content was reduced at higher temperature in the first season but there was no change in phenolic content in response to temperature treatments in either season. The concentrations of total amino acids at harvest increased in response to the higher temperature in the second season only. In addition, in the time course analysis of the second season, the accumulation of amino acids was increased at mid-ripening and ripening stage with the increased temperature. Significant qualitative changes in amino acid composition specifically the α-ketoglutarate family (i.e., glutamine, arginine, and proline) were found between the two temperatures. This study is the first to provide detailed analysis and quantification of individual amino acids and phenolics in Pinot noir in response to changes in temperature applied at veraison which could aid to develop adaptation strategies for viticulture in the future.

Large-scale phylogenomics of aquatic bacteria reveal molecular mechanisms for adaptation to salinity.

The crossing of environmental barriers poses major adaptive challenges. Rareness of freshwater-marine transitions separates the bacterial communities, but how these are related to brackish counterparts remains elusive, as do the molecular adaptations facilitating cross-biome transitions. We conducted large-scale phylogenomic analysis of freshwater, brackish, and marine quality-filtered metagenome-assembled genomes (11,248). Average nucleotide identity analyses showed that bacterial species rarely existed in multiple biomes. In contrast, distinct brackish basins cohosted numerous species, but their intraspecific population structures displayed clear signs of geographic separation. We further identified the most recent cross-biome transitions, which were rare, ancient, and most commonly directed toward the brackish biome. Transitions were accompanied by systematic changes in amino acid composition and isoelectric point distributions of inferred proteomes, which evolved over millions of years, as well as convergent gains or losses of specific gene functions. Therefore, adaptive challenges entailing proteome reorganization and specific changes in gene content constrains the cross-biome transitions, resulting in species-level separation between aquatic biomes.

Optimization of antimicrobial peptides for the application against biocorrosive bacteria

Microbiologically influenced corrosion is a common problem in the industrial field due to the deterioration of metals in the presence of various microorganisms, in particular sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB). A common method to reduce microbiologically influenced corrosion is the application of biocides. The limited number of suitable biocides and the resulting development of resistance, high dosage, and high application rate hinder an effective application. An environmentally friendly alternative could be the application of antimicrobial peptides (AMP), which have already been established in the field of medical devices for a while. Here, the successful treatment of different AMPs against 3 SRB and 1 SOB was demonstrated. The peptide L5K5W was favored due to its broad activity, high stability, and simple structure resulting in low synthesis costs. An alanine scan showed that substitution of leucine with tryptophan increased the activity of this peptide twofold compared to the original peptide against D. vulgaris, the main representative of SRB. Additional optimization of this modified peptide through changes in amino acid composition and lipidations significantly increased the effectiveness, finally resulting in a minimum inhibitory concentration (MIC) of 15.63 μg/mL against Desulfovibrio vulgaris. Even against the marine SRB Desulfovibrio indonesiensis with a required salt concentration of min. 2%, an activity of the peptides can be observed (MIC: 31.25 μg/mL). The peptides also remained stable and active for 7 days in the supernatant of the bacterial culture.Key points• Antimicrobial peptides provide an alternative to combat biocorrosive bacteria.• Optimization of the peptide sequence leads to a significant increase in activity.• The investigated peptides exhibit high stability, both in the medium and in the bacterial supernatant.Graphical abstract

Characterization of self-dyed silk yarn with Rhodamine B dye for fashion applications

PurposeThe textile industry has consumed large quantities of water and discharged large volumes of wastewater in the dyeing process. The study aims to characterize self-dyed silk with Rhodamine B (RhB) for fashion applications to reduce textile hazards to the environment and increase the added value of silk.Design/methodology/approachBombyx mori was fed with RhB-colored mulberry leaves (1500 ppm). The effects of self-dyeing were investigated via color strength K/S, Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscope, X-ray diffraction, tensile strength, color fastness to washing, rubbing, perspiration and light.FindingsSelf-dyed silk possesses effective coloration and impressive color fastness (4–5/5), higher crystalline index (CrI) (73.26 ± 2.28%), less thermal stability and tenacity, slight change in amino acid composition compared with the pristine and no existence of harmful aromatic azo amines and arylamine salts.Practical implicationsThe application of self-dyed silk with RhB dye has expanded new technology into fashion industry, contributing partly to economic growth and adding value to silk in the global supply chain. Besides, the self-dyeing will yield practical values in the reduction of dyeing discharge in textile industry.Originality/valueSelf-dyed silk was characterized for textile applications in comparison with pristine silk in terms of color strength and fastness as well as determined its polymeric properties relating to crystallinity, morphology, chemical composition, tensile properties and thermal stability which have not been investigated before.

Amino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis.

Amino acid compositions of cell culture media are empirically designed to enhance cell growth and productivity and vary both across media formulations and over the course of culture due to imbalance in supply and consumption. The interconnected nature of the amino acid transporters and metabolism suggests that changes in amino acid composition can affect cell physiology. In this study, we explore the effect of a step change in amino acid composition from a DMEM: F12-based medium to a formulation varying in relative abundances of all amino acids, evaluated at two amino acid concentrations (lean LAA vs. rich HAA). Cell growth was inhibited in LAA but not HAA. In addition to the expected effects on expression of the cell cycle, amino acid response and mTOR pathway genes in LAA, we observed an unanticipated effect on zinc uptake and efflux genes. This was accompanied by a lower tolerance to zinc supplementation in LAA but not in the other formulations. Histidine was sufficient but not necessary to prevent such zinc toxicity. Additionally, an unanticipated downregulation of genes in the cholesterol synthesis pathway was observed in HAA, accompanied by an increase in cellular cholesterol content, which may depend on the relative abundances of glutamine and other amino acids. This study shows that changes in the amino acid composition without any evident effect on growth may have profound effects on metabolism. Such analyses can help rationalize the designing of medium and feed formulations for bioprocess applications beyond replenishment of consumed components.

Surface modification of peanut meal with atmospheric cold plasma: Identifying the critical factors that affect functionality

SummaryPeanut meal (PM), a by‐product of processing, is hard to recycle due to its poor functional properties affected by the processing environment. To increase its utilisation value, atmospheric cold plasma (ACP) technology was used to enhance the functional properties of PM. Results showed that the soluble protein concentration, water holding capacity, surface hydrophobicity, foaming and emulsifying properties of PM were significantly improved after ACP treatment. The changes in amino acid composition, surface element content, apparent conformation, Fourier transform infrared spectroscopy and secondary structure of the protein in PM indicated that ACP treatment promoted the interaction between components of PM by introducing oxygen‐containing groups and producing etching. The protein molecule tended to be flexible, so as to improve the overall function of PM. ACP could be an effective and promising method to improve the functional properties of agricultural by‐products for further utilisation.

Hanseniaspora pseudoguilliermondii Improves the Flavor of Tilapia Fish Protein Hydrolysates

ABSTRACT To decrease the fishy odor of tilapia protein hydrolysates, this study investigated the flavor properties of fish protein hydrolysates (FPH) fermented by Hanseniaspora pseudoguilliermondii (FPHY), Lactobacillus plantarum 082 (FPHL) and combined fermentation (FPHLY). The thiobarbituric acid reactive substance (TBARS) values of FPHY (from 3.15 mg/L to 2.30 mg/L) and FPHL (from 2.89 mg/L to 1.29 mg/L) reduced by 26.98% and 55.36%, respectively. However, there were no obvious changes in amino acid composition. HS-SPME-GC-MS analysis showed that the main fishy components in FPH were hexanal and 1-octene-3-ol. Compared with FPHL, FPHY can produce more esters (1470.65 μg/mL), and the ester content was 352 times that of FPH (4.17 μg/mL) and 405 times that of FPHL (3.63 μg/mL). E-nose analysis, sensory evaluation and e-tongue analysis indicated that fermentation could decrease the bitterness of FPH, resulting in an improved taste. Therefore, H. pseudoguilliermondii fermentation could reduce the fishy odor and bitterness of FPH.

Crystal structure of SFPQ-NONO heterodimer

The Drosophila behavior/human splicing (DBHS) protein family is composed of the three members SFPQ, NONO and PSPC1. These proteins share a strong sequence and structural homology within the core-structured domains forming obligate homo- and heterodimers. This feature may lead to the simultaneous existence of six different dimeric complexes that sustain their function in many cellular processes such as pre-mRNA splicing, innate immunity, transcriptional regulation. In order to perform a complete structural analysis of all possible DBHS dimers, we have solved the crystal structure of the missing DBHS heterodimer SFPQ-NONO at 3.0 Å resolution. We identify subtle changes in amino acid composition and local secondary structure of the NOPS region orientation that may modulate affinity between complexes. Interestingly this area is found mutated in aggressive skin cancers and adenocarcinomas.

What can we learn from amino acids about oceanic organic matter cycling and degradation?

Abstract. Amino acids (AAs) mainly bound in proteins are major constituents of living biomass and non-living organic material in the oceanic particulate and dissolved organic matter pool. Uptake and cycling by heterotrophic organisms lead to characteristic changes in AA composition so that AA-based biogeochemical indicators are often used to elucidate processes of organic matter cycling and degradation. We analyzed particulate AA in a large sample set collected in various oceanic regions covering sinking and suspended particles in the water column, sediment samples, and dissolved AA from water column and pore water samples. The aim of this study was to test and improve the use of AA-derived biogeochemical indicators as proxies for organic matter sources and degradation and to better understand particle dynamics and interaction between the dissolved and particulate organic matter pools. A principal component analysis (PCA) of all data delineates diverging AA compositions of sinking and suspended particles with increasing water depth. A new sinking particle and sediment degradation indicator (SDI) allows a fine-tuned classification of sinking particles and sediments with respect to the intensity of degradation, which is associated with changes of stable isotopic ratios of nitrogen (δ15N). This new indicator is furthermore sensitive to sedimentary redox conditions and can be used to detect past anoxic early diagenesis. A second indicator emerges from the AA spectra of suspended particulate matter (SPM) in the epipelagic and that of the meso- and bathypelagic ocean and is a residence time indicator (RTI). The characteristic changes in AA patterns from shallow to deep SPM are recapitulated in the AA spectra of the dissolved organic matter (DOM) pool, so that deep SPM is more similar to DOM than to any of the other organic matter pools. This implies that there is equilibration between finely dispersed SPM and DOM in the deep sea, which may be driven by microbial activity combined with annealing and fragmentation of gels. As these processes strongly depend on physico-chemical conditions in the deep ocean, changes in quality and degradability of DOM may strongly affect the relatively large pool of suspended and dissolved AA in the ocean that amounts to 15 Pg amino acid carbon (AAC) and 89 ± 29 Pg AAC, respectively.

Investigating Phragmites australis response to copper exposure using physiologic, Fourier Transform Infrared and metabolomic approaches.

Phragmites australis (Cav.) Trin. ex Steud is a landscape plant with resistance to heavy metals that has significance in phytoremediation. However, little is known about the metabolomic background of the heavy metal resistance mechanisms of Phragmites . We studied copper stress on Phragmites and monitored physiological indicators such as malondialdehyde (MDA) and electrolyte leakage (EL). In addition, Fourier Transform Infrared (FTIR) was used to study the related chemical composition in the roots, stems, and leaves under copper stress. Furthermore, LC-MS technology was used to analyse the plants metabolic profile. Results showed that increased copper concentration in Phragmites led to the accumulation of MDA and EL. FTIR spectrum detected the presence of O-H and C=O stretching. O-H stretching was related to the presence of flavonoids, while C=O stretching reflected the presence of protein amide I. The latter was related to the change of amino acid composition. Both flavonoids and amino acids are regarded as contributors to the antioxidant of Phragmites under copper stress. Metabolomics analysis revealed that arginine and ayarin were accumulated and Phragmites leaves responded to copper stress with changes in the pool size of arginine and ayarin. It is speculated that they could improve resistance. Arginine is accumulated through two pathways: the citrulline decomposition and conversion pathway; and the circular pathway composed of ornithine, citrulline, l -argininosuccinate and arginine. Ayarin is synthesised through the quercetin methylation pathway. This study elucidates the antioxidant mechanisms for enhancing its resistance to heavy metal stress, thus improving of phytoremediation efficiency.

Effects of Dietary Protein Levels on Fecal Amino Acids Excretion and Apparent Digestibility, and Fecal and Ileal Microbial Amino Acids Composition in Weaned Piglets.

Background and Aims: The purpose of this study was to determine the effects of low protein diets with the same Lys, Met + Cys, Thr, and Trp levels as in high protein diets on the fecal amino acid excretion and apparent digestibility, and ileal and fecal microbial amino acids composition in weaned piglets.Methods: Fifty-four 21-day-old Duroc × Landrace × Yorkshire weaned piglets were randomly divided into three groups and fed with corn-soybean meal basal diets, in which the crude protein (CP) content was 20% (H-CP), 17% (M-CP), and 14% (L-CP), respectively. The experiment included a 7-day adaptation period and a 45-day trial period. Six piglets in each group were randomly slaughtered on days 10, 25, and 45 of the trial period, and the intestinal contents, intestinal mucosa, and feces were collected.Results: The results showed that the interaction between feeding time and dietary CP levels was reflected in the apparent digestibility of dietary CP and amino acid (AA) (p < 0.01). With the increase of age, the apparent digestibility of CP and AA were increased (p < 0.01). With the increase of CP levels, the excretion of nitrogen (N) was decreased (p < 0.01), whereas the flow of microbial AA in the ileum and feces were increased (p < 0.01). The interaction between feeding time and dietary CP levels was also reflected in the composition of AA in the ileum and stool of piglets (p < 0.01). The proportion of His, Lyr, Met, Cys, and Ser was lower than the average, whereas the proportion of Phe, Leu, Pro, Ala, Glu, and Asp was higher than the average. With the increase of age, the AA content of microorganisms increased (p < 0.01).Conclusion: All in all, this work revealed the changes of N, CP, and AA excretion and digestibility of feces and microorganisms of piglets under the combined action of different dietary protein levels and different feeding times, and also the changes of AA composition of intestinal microorganisms and AA composition of microorganisms.

Seasonal and Postharvest Changes in Amino Acid Composition in ‘Crimson Crisp’ Apple (Malus domestica Borkh.) in Response to Summer Foliar Urea Applications

Insufficient biologically available nitrogen (N) for yeast is a persistent issue facing cidermakers, whose apple juice base usually does not provide adequate nutrition for a complete fermentation. Cidermakers often supplement their juice with additional yeast assimilable nitrogen (YAN) in the cellar to aid fermentation. The development of biologically available N in apple juice is not well understood. In this study, juice samples from ‘Crimson Crisp®’ apples were taken at several sampling dates in the 2016, 2017, and 2018 growing seasons and analyzed for YAN using formol titration and high-performance liquid chromatography. It was observed that while the total YAN concentration in these apples drops from the period shortly after fruit set to the end of summer, YAN remains stable from several weeks before harvest until the date of harvest. The total YAN did not change after a 6-week postharvest storage period. By contrast, the individual amino acid components of YAN do change during this period. This experiment shows that foliar urea sprays in ‘Crimson Crisp®’ produce an increase in organic N in the juice, mostly in the form of asparagine. Increased organic N impacts yeast growth and sensory characteristics of cider and may be seen as desirable by cider producers.

Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases.

Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a noncatalytic residue in the WPD-loops of YopH and PTP1B result in shifted pH-rate profiles from an altered kinetic pKa of the nucleophilic cysteine. Compared to wild type, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations but suggest an increased preference for the WPD-loop-closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by nonchemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.