Chymotrypsin Inhibitor Research Articles

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2.

Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nanosecond and faster dynamics. Here we asked what effects the L49I and I57V substitutions, either individually or together, have on the slow conformational dynamics of CI2. We used 15 N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1 °C. As the temperature is increased the population of the excited state decreases. Structural changes in the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The substitutions in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the substituted residues and the well-ordered water molecules links subtle structural changes around the substituted residues to the region in the protein that experience slow conformational changes. This article is protected by copyright. All rights reserved.

Chymotrypsin inhibitor assay: Expressing, calculating, and standardizing inhibitory activity in absolute amounts of chymotrypsin inhibited

AbstractThere is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in soybean and other legume products, although trypsin inhibitor activity has been primarily measured. Recently, for accurately measuring CIA, an optimized method was developed in our laboratory and published elsewhere. Like most reported methods, the new method expresses CIA as chymotrypsin units (CU) inhibited (CUI)/mg sample. This arbitrary unit makes comparison of results impossible among different methods. The present study solved this problem by expressing CIA in absolute amounts of chymotrypsin inhibited (CId) and standardizing against a reference chymotrypsin. With the new method, two experiments were conducted, using four chymotrypsin reagents having different specific activities (SA), respectively. Experiment 1 determined the relationship between absorbance at 400 nm (A400) and μg chymotrypsin. Experiment 2 measured raw and heated soybeans, expressed CIA as CUI/mg sample and μg CId/mg sample, respectively, and determined conversion factors between the two units. Conversion factors determined in both experiments matched each other but varied with chymotrypsin SA. Yet, after standardizing against the reference chymotrypsin having a fixed SA of 150 N‐benzoyl‐L‐tyrosine‐p‐nitroanilide (BTNA) (equivalent to 50 N‐benzoyl‐L‐tyrosine ethyl ester [BTEE]) units/mg protein, a standardized conversion factor of 1.5 CUI (= 0.015 ΔA400) = 1 μg CId was determined. It remained consistent regardless of chymotrypsin SA, sample heating history, and levels. The new method can now express results in μg CId/mg sample (or mg/g) by dividing CUI/mg sample by 1.5. When other methods follow the same strategies for determining standardized conversion factors, CIA results could be comparable.

Biochemical composition, bioactivity, processing, and food applications of winged bean (Psophocarpus tetragonolobus): A review

AbstractThe winged bean, Psophocarpus tetragonolobus (L.) D.C., is called the four‐angled bean or Goa bean, which is an underutilized, non‐conventional, and multipurpose tropical legume cultivated in Southeast Asia and Papua New Guinea. The objective of this article was to reveal a comprehensive view on the nutritional properties, biochemical compositions, health benefits, processing, and food applications of winged beans. The average moisture, carbohydrate, protein, fat, crude fiber, and ash content of 138 genotypes of winged bean seeds were found as 9.09 ± 1.73%, 26.81 ± 6.88%, 34.98 ± 4.63%, 18.01 ± 2.27%, 10.24 ± 5.49%, and 4.16 ± 0.41%, respectively. Leaves of winged beans are good sources of vitamins such as vitamin C (14.5–128 mg/100 g), thiamine, riboflavin, niacin, vitamin B6, folate, vitamin A (5240–20,800 IU), and vitamin E. The seed of winged beans is rich in calcium, iron, phosphorus, zinc, and copper. The anti‐nutritional factors, namely, trypsin inhibitor, chymotrypsin inhibitor, phytic acid, saponin, tannin, oxalate, and flatulence saccharides of winged bean, were reported to be in the range of 40–99.5 TIU/mg of protein, 86.4–109.6 CIU/mg of protein, 4.09%–9.96%, 0.6%, 0.77%–0.97%, 0.5%, and 0.04%–0.18%, respectively. This bean can prevent diabetes, cancer, and asthma, boost immunity, and promote women's and men's reproductive health. Winged are transformed into consumable form by processing methods such as soaking, cooking, sprouting, fermentation, roasting, and so forth. This bean is processed into traditional products (curries, soup, pickles, etc.) and contemporary products (milk, tofu, tempeh, etc.). Future research should focus on promoting its utilization as an alternative to soybeans toward meeting global food and nutritional security.

How Sugars Protect Dry Protein Structure.

Extremotolerant organisms and industry exploit sugars as desiccation protectants, with trehalose being widely used by both. How sugars, in general, and the hydrolytically stable sugar trehalose, in particular, protect proteins is poorly understood, which hinders the rational design of new excipients and implementation of novel formulations for preserving lifesaving protein drugs and industrial enzymes. We employed liquid-observed vapor exchange nuclear magnetic resonance (LOVE NMR), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA) to show how trehalose and other sugars protect two model proteins: the B1 domain of streptococcal protein G (GB1) and truncated barley chymotrypsin inhibitor 2 (CI2). Residues with intramolecular H-bonds are most protected. The LOVE NMR and DSC data indicate that vitrification may be protective. Combining LOVE NMR and TGA data shows that water retention is not important. Our data suggest that sugars protect protein structure as they dry by strengthening intraprotein H-bonds and water replacement and that trehalose is the stress-tolerance sugar of choice because of its covalent stability.

Production of mycelial biomass, proteases and protease inhibitors by Ganoderma lucidum under different submerged fermentation conditions.

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.

Paper-based flow sensor for detection of chymotrypsin and its inhibitors via viscosity change of gelatin

Chymotrypsin (CHT) is an important proteolytic enzyme that can quickly decompose denatured proteins. Herein, we reported a new, simple, rapid method for the detection of CHT and its inhibitors by a paper-based flow sensor for the first time. The principle of the sensor is the interconversion of solidification and dissolution of gelatin. Generally, the 3D structure of gelatin can be enzymatically hydrolyzed by CHT releasing internal water, leading to a gradual increase in the water mark coverage area. Conversely, water cannot be released and the water mark coverage area is 0 in the absence of CHT, which is attributed to the water being trapped in the gelatin hydrogel. The limit of detection of CHT is determined to be 0.8 U/mL. In addition, the detection of CHT in milk, screening of CHT inhibitors and determination of Bowman-Birk inhibitors in soymilk were successfully achieved. Interestingly, the sensor only requires gelatin hydrogel, pH indicator strips, and PVC plates. In summary, a rapid, high-throughput, low-cost strategy for the detection of CHT and screening of CHT inhibitors was developed. Moreover, this method also allows the detection of CHT in non-transparent samples such as milk and soymilk. Paper-based flow sensors are highly promising for applications in food quality control, new medicine discovery and disease diagnosis, which also have a high potential for commercialization.

Selection of Lentil (Lens Culinaris (Medik.)) Genotypes Suitable for High-Temperature Conditions Based on Stress Tolerance Indices and Principal Component Analysis.

Legumes, including lentil, are a valuable source of carbohydrates, fiber, protein and vitamins and minerals. Their nutritional characteristics have been associated with a reduction in the incidence of various cancers, HDL cholesterol, type 2 diabetes and heart disease. Among these quality parameters, lectins have been associated with reducing certain forms of cancer, activating innate defense mechanisms and managing obesity. Protease inhibitors such as trypsin and chymotrypsin inhibitors have been demonstrated to reduce the incidence of certain cancers and demonstrate potent anti-inflammatory properties. Angiotensin I-converting enzyme (ACE) inhibitor has been associated with a reduction in hypertension. Therefore, legumes, including lentils, should be part of our daily food intake. However, high temperatures at the terminal stage is a major abiotic constraint leading to a reduction in lentil yield and seed quality. Thus, the selection of heat-tolerant genotypes is essential to identifying the potential for high yields with stable performance. To select lentil genotypes, an experiment was conducted with 60 genotypes including local landraces, advanced breeding lines, commercial varieties and exotic germplasm under stress and non-stress conditions from 2019 to 2020. This study was followed by a subset study involving screening based on a few physicochemical parameters and reproductive traits along with field performances. Different tolerance indices (i.e., stress susceptible index (SSI), relative heat index (RHI), tolerance (TOL), mean productivity (MP), stress tolerance index (STI), geometric mean productivity (GMP), yield index (YI), yield stability index (YSI), heat-resistance index (HRI), modified stress-tolerance index (MSTI), abiotic tolerance index (ATI) and stress susceptibility percentage (SSPI)) were used for the selection of the genotypes along with field performance. Biplot analysis was further performed for choosing the most suitable indices. Based on principal components analysis, the GMP, MP, RRI, STI, YI, YSI, ATI and MSTI indices were identified as the most reliable stress indicators, and these indicators might be used for distinguishing heat-tolerant genotypes. Based on the stress indices, the genotypes BLX 05002-3, BLX 10002-20, LRIL-21-1-1-1-1, LRIL-21-1-1-1-1-6 and BLX 09015 were selected as the most stable and heat-tolerant genotypes. In contrast, the genotypes LG 198, Bagura Local, BLX 0200-08-4, RL-12-178, Maitree, 91517 and BLX 11014-8 were selected as the most heat sensitive. Data also exhibited an average yield reduction of 59% due to heat stress on the lentils. Moreover, eight heat-tolerant (HT) genotypes (BLX 09015, PRECOZ, LRL-21-112-1-1-1-1-6, BLX 05002-3, LR-9-25, BLX 05002-6, BARI Masur-8 and RL-12-181), and two heat-susceptible (HS) genotypes (BLX 12009-6, and LG 198) were selected from the screened genotypes and subjected to further analysis by growing them in the following year under similar conditions to investigate the mechanisms associated with heat tolerance. Comparative studies on reproductive function and physiochemical traits revealed significantly higher pollen viability, proline accumulation, relative water content, chlorophyll concentration and a lower membrane stability index in HT genotypes under heat stress. Therefore, these heat-tolerant genotypes could be used as the parents in the hybridization program for achieving heat-tolerant transgressive segregation.

Exploring ex vivo peptideolysis of thymopentin and lipid-based nanocarriers towards oral formulations

The oral delivery of medicines is the most popular route of administration for patients. However, thymopentin (TP5) is only available in the market in forms for parenteral administration. In large part, this is because of extensive peptidolytic degradation in the gastrointestinal tract (GIT), which decreases the amount of TP5 available for absorption. This study aims to understand the extent of TP5 peptideolysis and determine effective inhibitors and suitable lipid-based nanocarriers to aid in the development of an effective oral delivery formulation. Enzymatic degradation kinetics of TP5 was investigated in the presence or absence of mucosal and luminal components extracted from various parts of the rat intestine, including the duodenum, jejunum, ileum, and colon. Inhibition of TP5 enzymatic peptidolysis was screened in the presence or absence of EDTA, trypsin and chymotrypsin inhibitors from soybean (SBTCI), and bestatin. TP5 with SBTCI was loaded into lipid-based nanocarriers, including microemulsions, niosomes and solid lipid nanoparticles. These TP5-loaded nanocarriers were investigated through characterization of morphology, particle size, zeta potential, entrapment efficacy (EE%), and ex vivo rat intestinal degradation studies to select a lead formulation for a future oral drug delivery study. The degradation kinetics of TP5 followed pseudo-first-order kinetics, and the biological metabolism of TP5 was displayed in the presence of luminal contents, indicating that TP5 is sensitive to luminal enzymes. Notably, a considerable decrease in TP5 peptidolysis was found in the presence of SBTCI, bestatin, and EDTA. TP5 and SBTCI were loaded into three lipid-based delivery systems, displaying superior protection under ex vivo intestinal luminal contents and mucosal homogenates for 6 h compared with the pure drug solution. These findings suggest that using select inhibitors and lipid-based nanocarriers can decrease peptide degradation and may improve oral bioavailability of TP5 following oral administration.

The effect of radiofrequency heat treatment on trypsin inhibitor activity and in vitro digestibility of soybean varieties (Glycine max. (L.) Merr.)

Kunitz (KTI) and Bowman-Birk (BBI) trypsin inhibitors were characterized in soybean seeds. Cultivars having KTI/BBI (Pannónia Kincse, PK) or lacking KTI (Aries; Hilario; Bahia) were assessed with well-characterized soybean varieties having Ti-a or ti types of KTI mobility. The TIA values of Pannónia Kincse (9.8 ± 0.48 mg/g) were not significantly different (p ≤ 0.05) from Ti-a samples (10.07 ± 1.86 mg/g), while of Aires, Bahia, Hilario (6.19 ± 1.89) were identical (p ≤ 0.05) with ti samples (6.63 ± 1.99). Radiofrequency heat treatment (RF) decreased TIA values (p ≤ 0.05) at ≥ 100 °C. However, in the traditional soybean variety, the RF at 110 °C was more effective in eliminating the residual KTI activity. The remaining or the disapperaing bioactive form of trypsin inhibitors were succesfully characterized by the means of a standardized in vitro digestion model. It showed that residual BBI-originated trypsin inhibitor activity was in the stomach even after RF at 110 °C, whereas its chymotrypsin inhibitor activity was not detectable at all. Although PK and KTI null types of soybean seeds still required an energy-saving, gentle heat treatment to inactive the trypsin inhibitors before using them as food or feed, the physicochemical properties and processing quality of soybean products were protected, improved.

Method development and optimization for measuring chymotrypsin and chymotrypsin inhibitor activities.

Protease inhibitors of protein nature are rich in seeds of legume crops. There are two common types: Kunitz inhibitor, which mainly inhibits trypsin, and Bowman-Birk inhibitor, which inhibits both trypsin and chymotrypsin. Historically, trypsin inhibitor activity in legume products has been of primary interest for measurement. However, as plant proteins are increasingly used for food or feed in recent years, there is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in these products as well. Reported methods for CIA assay vary greatly and are incompletely described. No standardized or official method is available. The present study focused on developing a robust method for accurately measuring CIA, using N-benzoyl-L-tyrosine p-nitroanilide (BTpNA) as a substrate. Since BTpNA is not water soluble, a water-miscible organic solvent must be present. After investigating the effects of several factors, such as absorption spectra, organic solvent type and concentration, substrate and enzyme concentrations, inhibitor levels (which affected % chymotrypsin inhibition), the sequence of adding reagents, extractant and extraction time, and so forth, an optimized method for CIA measurement was finally developed. It features dimethylformamide as the organic solvent, the enzyme-last sequence, 5ml total assay volume, and calculation of the inhibitor activity based on 40% chymotrypsin inhibition. The method can also be slightly modified for measuring chymotrypsin activity. The robust performance of the method was verified by measuring 11 assorted protein products, paving a way for standardization. PRACTICAL APPLICATION: With an increasing use of plant proteins, there is an urgent need to measure chymotrypsin inhibitor activity in various protein products with accuracy. After thoroughly investigating several factors, an optimized method for measuring chymotrypsin inhibitor activity in various protein products was developed. The proposed method is sensitive and robust, providing a basis for standardization. It can also be used for measuring chymotrypsin activity.

Ability of (extruded) pea protein products to partially replace pork meat in emulsified cooked sausages

The effect of replacing 20% pork meat in cooked sausages by Pea Protein Isolate (PPI), pea Low Moisture Extrudates (LME), and pea High Moisture Extrudate (HME) on the nutritional, techno-functional and sensorial properties of emulsified cooked sausages was investigated. All hybrid sausages contained all essential amino acids in sufficient amounts. Interestingly, extrusion significantly reduced the Anti-Nutritional Factors (ANF) trypsin and chymotrypsin inhibitors, and the allergenic pea Pis s2 (convicilin) content. Inclusion of (texturized) pea proteins into hybrid meat sausages resulted in weaker networks, and thus (significant) softer bite, and significant color changes. The expert taste panel did not note any significant differences between the hybrid sausage with PPI and its reference for all sensorial attributes. Industrial relevanceProducing meat substitutes with powdered or extruded plant-based protein products, such as PPI, LME, and HME, can result in several limitations concerning textural, nutritional and sensorial features. To avoid these hurdles, hybrid meat products, where a fraction of the meat is replaced by alternative proteins, may bring a solution.This work demonstrated that 20% of pork meat can be replaced by pea products PPI, LME or HME in emulsified cooked sausages without any implications towards the amino acid profile. Moreover, using extruded pea products could even significantly reduce ANF trypsin and chymotrypsin inhibitors and the allergenic pea convicilin contents. This knowledge plays a crucial role for product development, as this demonstrates that hybrid meats can be produced with great nutritional qualities. The challenges identified were that the hybrid sausage exhibited a weaker network with softer texture, and the inclusion of extruded proteins led to large cavities with jelly-like excretion. This demonstrates the importance of selecting suitable (forms of) plant proteins for the production of hybrid meats with great techno-functional and organoleptic properties.

Physicochemical Properties and Elimination of the Activity of Anti-Nutritional Serine Protease Inhibitors from Mulberry Leaves.

Mulberry leaf is an excellent protein resource that can be used as feed additive for livestock and poultry. Nevertheless, the use of mulberry leaves in animal diets is limited by its protease inhibitors, tannic acid and other anti-nutritional factors. This study systematically analyzed the type and activity of serine protease inhibitors (SPIs) from the leaves of 34 mulberry varieties, aiming to reveal the physicochemical properties and inactivation mechanism of SPIs. The types and activities of trypsin inhibitors (TIs) and chymotrypsin inhibitors (CIs) exhibited polymorphisms among different mulberry varieties. The highest number of types of inhibitors was detected in Jinshi, with six TIs (TI-1~TI-6) and six CIs (CI-1~CI-6). TIs and CIs exhibited strong thermal and acid–base stability. High-temperature and high-pressure treatment could reduce the activities of TIs and CIs to a certain extent. β-mercaptoethanol treatment could completely abolish TIs and CIs, suggesting that the disulfide bridges were critical for their inhibitory activities. The Maillard reaction could effectively eliminate the inhibitory activities of TI-1~TI-4 and CI-1~CI-4. This study reveals the physicochemical properties and inactivation mechanisms of the anti-nutritional SPIs from mulberry leaves, which is helpful to exploit mulberry-leaf food with low-activity SPIs, promote the development and utilization of mulberry-leaf resources in animal feed and provide reference for mulberry breeding with different functions.

More Light Please: Daphnia Benefit From Light Pollution by Increased Tolerance Toward Cyanobacterial Chymotrypsin Inhibitors

Cryptochromes are evolutionary ancient blue-light photoreceptors that are part of the circadian clock in the nervous system of many organisms. Cryptochromes transfer information of the predominant light regime to the clock which results in the fast adjustment to photoperiod. Therefore, the clock is sensitive to light changes and can be affected by anthropogenic Artificial Light At Night (ALAN). This in turn has consequences for clock associated behavioral processes, e.g., diel vertical migration (DVM) of zooplankton. In freshwater ecosystems, the zooplankton genus Daphnia performs DVM in order to escape optically hunting predators and to avoid UV light. Concomitantly, Daphnia experience circadian changes in food-supply during DVM. Daphnia play the keystone role in the carbon-transfer to the next trophic level. Therefore, the whole ecosystem is affected during the occurrence of cyanobacteria blooms as cyanobacteria reduce food quality due to their production of digestive inhibitors (e.g., protease inhibitors). In other organisms, digestion is linked to the circadian clock. If this is also the case for Daphnia, the expression of protease genes should show a rhythmic expression following circadian expression of clock genes (e.g., cryptochrome 2). We tested this hypothesis and demonstrated that gene expression of the clock and of proteases was affected by ALAN. Contrary to our expectations, the activity of one type of proteases (chymotrypsins) was increased by ALAN. This indicates that higher protease activity might improve the diet utilization. Therefore, we treated D. magna with a chymotrypsin-inhibitor producing cyanobacterium and found that ALAN actually led to an increase in Daphnia’s growth rate in comparison to growth on the same cyanobacterium in control light conditions. We conclude that this increased tolerance to protease inhibitors putatively enables Daphnia populations to better control cyanobacterial blooms that produce chymotrypsin inhibitors in the Anthropocene, which is defined by light pollution and by an increase of cyanobacterial blooms due to eutrophication.

Insect protease inhibitors; promising inhibitory compounds against SARS-CoV-2 main protease

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has adversely affected global health since its emergence in 2019. The lack of effective treatments prompted worldwide efforts to immediately develop therapeutic strategies against COVID-19. The main protease (Mpro) of SARS-CoV-2 plays a crucial role in viral replication, and therefore it serves as an attractive target for COVID-19-specific drug development. Due to the richness and diversity of insect protease inhibitors, we docked SARS-CoV-2 Mpro onto 25 publicly accessible insect-derived protease inhibitors using the ClusPro server, and the regions with high inhibitory potentials against Mpro were used to design peptides. Interactions of these inhibitory peptides with Mpro were further assessed by two directed docking programs, AutoDock and Haddock. AutoDock analysis predicted the highest binding energy (−9.39 kcal/mol) and the lowest inhibition constant (130 nM) for the peptide 1KJ0-7 derived from SGCI (Schistocerca gregaria chymotrypsin inhibitor). On the other hand, Haddock analysis resulted in the discovery of a different peptide designated 2ERW-9 from infestin, a serine protease inhibitor of Triatoma infestans, with the best docking score (−131), binding energy (−11.7 kcal/mol), and dissociation constant (2.6E-09 M) for Mpro. Furthermore, using molecular dynamic simulations, 1KJ0-7 and 2ERW-9 were demonstrated to form stable complexes with Mpro. The peptides also showed suitable drug-likeness properties compared to commercially available drugs based on Lipinski's rule. Our findings present two peptides with possible protease inhibitor activities against Mpro and further demonstrate the potential of insect-derived peptides and computer-aided methods for drug discovery.

Double Mutant of Chymotrypsin Inhibitor 2 Stabilized through Increased Conformational Entropy.

The conformational heterogeneity of a folded protein can affect not only its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here, we have examined whether changed native-state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics of amide groups in the backbone and the methyl groups in the side chains with molecular dynamics simulations to quantify the native-state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: a reduction in conformational dynamics (and entropy estimated from this) of the native state of the L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native-state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

Variable-Temperature Native Mass Spectrometry for Studies of Protein Folding, Stabilities, Assembly, and Molecular Interactions.

The structures and conformational dynamics of proteins, protein complexes, and their noncovalent interactions with other molecules are controlled specifically by the Gibbs free energy (entropy and enthalpy) of the system. For some organisms, temperature is highly regulated, but the majority of biophysical studies are carried out at room, nonphysiological temperature. In this review, we describe variable-temperature electrospray ionization (vT-ESI) mass spectrometry (MS)-based studies with unparalleled sensitivity, dynamic range, and selectivity for studies of both cold- and heat-induced chemical processes. Such studies provide direct determinations of stabilities, reactivities, and thermodynamic measurements for native and non-native structures of proteins and protein complexes and for protein-ligand interactions. Highlighted in this review are vT-ESI-MS studies that reveal 40 different conformers of chymotrypsin inhibitor 2, a classic two-state (native → unfolded) unfolder, and thermochemistry for a model membrane protein system binding lipid and its regulatory protein.

The Kunitz chymotrypsin inhibitor from Erythrina velutina seeds displays activity against HeLa cells through arrest in cell cycle.

Erythrina velutina is a species of arboreal leguminous that occurs spontaneously in the northeastern states of Brazil. Leguminous seeds represent an abundant source of peptidase inhibitors, which play an important role in controlling peptidases involved in essential biological processes. The aim of this study was to purify and characterize a novel Kunitz-type peptidase inhibitor from Erythrina velutina seeds and evaluate its anti-proliferative effects against cancer cell lines. The Kunitz-type chymotrypsin inhibitor was purified from Erythrina velutina seeds (EvCI) by ammonium sulphate fractionation, trypsin- and chymotrypsin-sepharose affinity chromatographies and Resource Q anion-exchange column. The purified EvCI has a molecular mass of 18kDa with homology to a Kunitz-type inhibitor. Inhibition assays revealed that EvCI is a competitive inhibitor of chymotrypsin (with K i of 4 × 10-8M), with weak inhibitory activity against human elastase and without inhibition against trypsin, elastase, bromelain or papain. In addition, the inhibitory activity of EvCI was stable over a wide range of pH and temperature. Disulfide bridges are involved in stabilization of the reactive site in EvCI, since the reduction of disulfide bridges with DTT 100mM abolished ~ 50% of its inhibitory activity. The inhibitor exhibited selective anti-proliferative properties against HeLa cells. The incubation of EvCI with HeLa cells triggered arrest in the cell cycle, suggesting that apoptosis is the mechanism of death induced by the inhibitor. EvCI constitutes an interesting anti-carcinogenic candidate for conventional cervical cancer treatments employed currently. The EvCI cytostatic effect on Hela cells indicates a promised compound to be used as anti-carcinogenic complement for conventional cervical treatments employed currently.

Mating Stimulates the Immune Response and Sperm Storage-Related Genes Expression in Spermathecae of Bumblebee (Bombus terrestris) Queen.

Bumblebee queens have remarkable spermathecae that store sperm for year-round reproduction. The spermathecal gland is regarded as a secretory organ that could benefit sperm storage. Queen mating provokes substantial physiological, behavioral, and gene expression changes. Here, the transcriptomes of spermathecae were compared between virgins and mated queens of the bumblebee, Bombus terrestris L., at 24 h post mating. Differentially expressed genes were further validated by real time quantitative PCR and immunofluorescence assay. In total, the expression of 11, 069 and 10, 862 genes were identified in virgins and mated queens, respectively. We identified that 176 differentially expressed genes between virgin and mated queen spermathecae: 110 (62.5%) genes were upregulated, and 66 (37.5%) genes were downregulated in mated queens. Most of the differentially expressed genes validated by RT-qPCR were concentrated on immune response [i.e., leucine-rich repeat-containing protein 70 (35.8-fold), phenoloxidase 2 (41.9-fold), and defensin (4.9-fold)] and sperm storage [i.e., chymotrypsin inhibitor (6.2-fold), trehalose transporter Tret1 (1.7-, 1.9-, 2.4-, and 2.4-fold), and heterogeneous nuclear ribonucleoprotein A3 (1.2-, and 2.6-fold)] functions in the spermathecae of mated queens. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) was hypothesized to promote the mating behavior according to RT-qPCR and immunofluorescence assay. The expression levels of most upregulated immune genes were decreased significantly at 3 days post mating. In conclusion, the external sperm transfer into spermathecae led to the significantly upregulated immune response genes in bumblebees. These gene expression differences in queen spermathecae contribute to understanding the bumblebee post mating regulatory network.

Protection by desiccation-tolerance proteins probed at the residue level.

Extremotolerant organisms from all domains of life produce protective intrinsically disordered proteins (IDPs) in response to desiccation stress. In vitro, many of these IDPs protect enzymes from dehydration stress better than U.S. Food and Drug Administration-approved excipients. However, as with most excipients, their protective mechanism is poorly understood. Here, we apply thermogravimetric analysis, differential scanning calorimetry, and liquid-observed vapor exchange (LOVE) NMR to study the protection of two model globular proteins (the B1 domain of staphylococcal protein G [GB1] and chymotrypsin inhibitor 2 [CI2]) by two desiccation-tolerance proteins (CAHS D from tardigrades and PvLEA4 from an anhydrobiotic midge), as well as by disordered and globular protein controls. We find that all protein samples retain similar amounts of water and possess similar glass transition temperatures, suggesting that neither enhanced water retention nor vitrification is responsible for protection. LOVE NMR reveals that IDPs protect against dehydration-induced unfolding better than the globular protein control, generally protect the same regions of GB1 and CI2, and protect GB1 better than CI2. These observations suggest that electrostatic interactions, charge patterning, and expanded conformations are key to protection. Further application of LOVE NMR to additional client proteins and protectants will deepen our understanding of dehydration protection, enabling the streamlined production of dehydrated proteins for expanded use in the medical, biotechnology, and chemical industries.

Protein simulation in supercritical CO2: The challenge of force field

Supercritical CO2 is one of the most important green solvents for biotechnological applications. Due to the high-pressure conditions and gas–liquid properties of the fluid, the structural investigations of the biological macromolecules such as proteins and enzymes in supercritical conditions is problematic. Molecular dynamics simulation has emerged as a good tool to study the molecular-levels phenomena in macromolecule/supercritical CO2 environments. Many unsolved aspects of such systems have been explored by this method in recent years. One of the most important challenges in biomolecular simulations in supercritical CO2 is the applied force field for the bio-macromolecules. Most of simulations in supercritical CO2 have been performed by united-atoms and GROMOS96-based force fields for proteins and enzymes. The reliability of the obtained results should be examined by applying the other protein force fields. There is no comprehensive work about the effect of the force field on a single protein model in supercritical CO2. In this work, I have applied four famous biomolecular force fields including: GROMOS96 (43a1), CHARMM (27), AMBER (03) and OPLS-AA for simulation of chymotrypsin inhibitor 2 as a model protein in supercritical CO2 and also in the aqueous conditions. Different structural properties of protein have been examined using the different force fields. Results show that all of the force fields confirm the structural deviations of the protein structure from its native state in supercritical CO2. GROMOS43a1, similar to the previous simulations, showed the higher flexibility of protein backbone in scCO2 in comparison to the aqueous condition. In contrast, in simulations with all-atom force fields, the protein flexibility was almost identical in two solvents. Simulations by all of the force fields confirm the previously proposed mechanism for protein denaturation in scCO2 which states that the changing of the interaction pattern of protein destabilizes the conformation in supercritical CO2. However, the extent of changes was different among the force fields. While united-atom force field show higher changes and deviations from the native state, all-atom force fields, in agreement with each other, show moderate changes and deviations.