Thermal Denaturation Research Articles

Effect of ultrasonic treatment on enzymes: Decoupling the relation between the ultrasonic driven conformational change and enzyme activity

Considering the growing interest in enzyme-based extraction technology as a safe and eco-friendly extraction technique, along with the relatively high cost associated with enzymatic applications, it became necessary to explore novel strategies aimed to improve enzyme activity. In this study, the impact of ultrasonic treatment on commercial cellulase and pectinase was investigated. As this effect may be influenced by various ultrasonic and enzyme-related parameters, changes in enzyme conformation were explored under optimal and non-optimal enzyme conditions. The intrinsic fluorescence spectrum was utilized as a tool for monitoring these changes. Additionally, the enzyme’s catalytic potential was also assessed under the same conditions. Results indicated that the impact of ultrasonic treatment on enzyme conformation primarily depends on the total ultrasonic energy delivered to the system, rather than other ultrasonic parameters such as power, sample volume, treatment time, or duty cycle. The maximum relative decrease in intrinsic fluorescence intensity of Pectinex® Ultra Clear (PUC) and Pectinex® Ultra SPL (PUS) after ultrasonic treatment was approximately 51% and 55%, respectively, while the decrease induced by thermal denaturation was 25% and 30% respectively. Furthermore, a blue shift in the fluorescence spectrum of both pectinases was observed upon sonication for all process conditions indicating a change in enzyme conformation. However, ultrasonic treatment did not result in a significant change in enzyme activity, suggesting that these conformational adjustments may occur in regions other than the active sites. Moreover, ultrasonicated pectinases and cellulases did not exhibit any improvement in their catalytic potential under either optimal or non-optimal conditions.

Interacting Characteristics of Chrysene with Free DNA in vitro

Deoxyribonucleic acid (DNA), an important material in life science, is considered a natural and highly specific functional biopolymer. Various analytical techniques were used to analyze the interaction of chrysene with free DNA in vitro, and based on this interaction, we studied the removal of chrysene from contaminated water by DNA via magnetic beads-based separation. The results of UV-visible spectroscopy, DNA thermal denaturation measurement, circular dichroism (CD) spectroscopy, and fluorescence microscopy showed that chrysene interacted with DNA through an intercalating mode. The results of fluorescence spectroscopy demonstrated that DNA had a static quenching effect on chrysene, indicating the formation of a stable complex between chrysene and DNA. Thermodynamic studies revealed that the interaction of chrysene with free DNA was spontaneous in vitro. In addition, chrysene could be efficiently removed from contaminated water by DNA via DNA-intercalation and magnetic bead-based separation. The chrysene removal efficiency of DNA was better than that of activated carbon even when the amount of activated carbon was 400 times more than that of DNA. The results of this study provide insights into the development of new chrysene elimination processes based on DNA-intercalation in water pollution.

Comparison of Conformations and Interactions with Nicotinic Acetylcholine Receptors for E. coli-Produced and Synthetic Three-Finger Protein SLURP-1

SLURP-1 is a three-finger human protein targeting nicotinic acetylcholine receptors (nAChRs). The recombinant forms of SLURP-1 produced in E. coli differ in added fusion fragments and in activity. The closest in sequence to the naturally occurring SLURP-1 is the recombinant rSLURP-1, differing by only one additional N-terminal Met residue. sSLURP-1 can be prepared by peptide synthesis and its amino acid sequence is identical to that of the natural protein. In view of recent NMR analysis of the conformational mobility of rSLURP-1 and cryo-electron microscopy structures of complexes of α-bungarotoxin (a three-finger snake venom protein) with Torpedo californica and α7 nAChRs, we compared conformations of sSLURP-1 and rSLURP-1 by Raman spectroscopy and CD-controlled thermal denaturation, analyzed their competition with α-bungarotoxin for binding to the above-mentioned nAChRs, compared the respective receptor complexes with computer modeling and compared their inhibitory potency on the α9α10 nAChR. The CD revealed a higher thermostability of sSLURP-1; some differences between sSLURP-1 and rSLURP-1 were observed in the regions of disulfides and tyrosine residues by Raman spectroscopy, but in binding, computer modeling and electrophysiology, the proteins were similar. Thus, sSLURP-1 and rSLURP-1 with only one additional Met residue appear close in structure and functional characteristics, being appropriate for research on nAChRs.

Dark nanodiscs for evaluating membrane protein thermostability by differential scanning fluorimetry

Measuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed nonfluorescent membrane scaffold protein to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the “dark” nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs loaded with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) lipids show a complex biphasic thermal unfolding pattern with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5°C and 77.5°C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a DMPC and DMPG nanodisc. To extend the utility of this method, we evaluate and compare the thermostability of DsbB in different lipid environments. We introduce this method as a new tool that can be used to understand how compositionally and biophysically complex lipid environments drive membrane protein stability.

Physicochemical, functional, and antioxidant properties of black soldier fly larvae protein.

This study explores the multifaceted attributes of black soldier fly larvae protein (BSFLP), focusing on its physicochemical, functional, and antioxidant properties. BSFLP is characterized by 16 amino acids, with a predominant random coil secondary structure revealed by circular dichroism spectra. Differential scanning calorimetry indicates a substantial thermal denaturation temperature of 97.63°C. The protein exhibits commendable solubility, emulsification, and foaming properties in alkaline and low-salt environments, albeit with reduced water-holding capacity and foam stability under elevated alkaline and high-temperature conditions. In vitro assessments demonstrate that BSFLP displays robust scavenging proficiency against 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and hydroxyl radicals, with calculated EC50 values of 1.90±0.57, 0.55±0.01, and 1.14±0.02mg/mL, respectively, along with notable reducing capabilities. Results from in vivo antioxidant experiments reveal that BSFLP, administered at doses of 300 and 500mg/kg, significantly enhances the activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) (p<0.05) while simultaneously reducing malondialdehyde levels in both serum and tissues of d-galactose-induced oxidative stress in mice. Moreover, the protein effectively attenuates oxidative damage in liver and hippocampal tissues. These findings underscore the potential utility of BSFLP as a natural antioxidant source, with applications spanning the food, pharmaceutical, and cosmetic industries. PRACTICAL APPLICATION: Black soldier fly larvae protein emerges as an environmentally sustainable reservoir of natural antioxidants, holding significant promise for the food, pharmaceutical, and cosmetic sectors. Its advantageous amino acid composition, robust thermal resilience, and impressive functional attributes position it as a compelling subject for continued investigation and advancement in various applications.

Impact of heat-set and cold-set gelling polysaccharides on potato protein gelation: Gellan gum, agar, and methylcellulose

The plant-based meat market is growing due to concerns about the sustainability of the modern food system. Plant-based meats are often constructed from blends of food biopolymers, such as plant proteins and polysaccharides. This study characterizes the thermal setting properties of potato protein (10%) and polysaccharide (2 wt%) mixtures containing polysaccharides with different reversible gelling behaviors: agar (cold set); gellan gum (cold set); or methyl cellulose (heat set). Each sample was heated above the thermal denaturation temperature of the potato proteins to promote protein unfolding, aggregation, and network formation. The addition of the polysaccharides tended to increase the complex shear modulus of the biopolymer blends before heating but increase or decrease it after heating depending on polysaccharide type. The shear modulus and hardness of the gels formed after heating tended to increase after addition of the cold-set polysaccharides but decrease after the addition of the heat-set polysaccharides, especially at refrigerator temperatures. Differential scanning calorimetry data showed that the potato proteins underwent irreversible denaturation after heating for all samples. Zeta-potential analysis showed that the potato proteins and all polysaccharides were negatively charged under the pH conditions used. There was greater water loss during heating for the composite gels containing methyl cellulose, which may have been due to its increase hydrophobicity at high temperatures. Appearance and microstructure analysis showed that phase-separation occurred after mixing the polysaccharides with the potato proteins, which was attributed to a segregation mechanism. The knowledge obtained from this study may help the food industry to optimize protein-polysaccharide blends to create gels with characteristics and structures that can mimic different plant-based meat products.

Investigating thermal denaturation kinetics of 7S and 11S proteins and lipoxygenase in soymilk through ohmic heating

AbstractThe purpose of this study is to investigate denaturation kinetics of 7S and 11S proteins as well as lipoxygenase in soymilk during ohmic heating treatment. Because denaturation kinetics of 7S and 11S are nth‐order reaction, a reaction order was estimated based on experimental data and simulation results using Arrhenius parameters calculated through differential scanning calorimetry dynamic method. Denaturation kinetic of lipoxygenase was determined by denaturation ratio which estimated based on oxygenase activity of its after ohmic heating treatment at 65, 70, and 72.5°C. The reaction orders of 7S and 11S proteins during ohmic heating of raw soymilk were determined as 3.25 and 1.52, respectively; while the reaction order of 11S protein in preheated soymilk at 75°C for 1 h was found to be 1.94 with a good correlation between experimental data and simulation result. Electrophoresis results indicated that trypsin inhibitor might affect the denaturation of 11S protein during ohmic heating. The denaturation of lipoxygenase during ohmic heating followed a first‐order reaction at all temperatures, and denaturation simulation using the Arrhenius parameters corresponded well with the experimental data.Practical ApplicationsThis study provides valuable insights into improving a quality of soymilk products. 7S and 11S proteins play a critical role in determining viscosity of soymilk and tofu made from soymilk, which together account for a large proportion of the total proteins in soymilk. Understanding the thermal denaturation kinetics of 7S and 11S proteins can help food manufacturers optimize their processing parameters to achieve the desired physical properties of soymilk. Moreover, lipoxygenase is responsible for producing beany flavor components in soymilk, and understanding its thermal denaturation kinetic can lead to production of soymilk with high consumer acceptance. Our research supports a practical application of ohmic heating technology in the processing of soymilk.

Investigation on CT-DNA and Protein Interaction of New Pd(II) Complexes Involving Ceftazidime and 3-Amino-1,2,3-triazole: Synthesis, Characterization, Biological Impact, Anticancer Evaluation, and Molecular Docking Approaches.

Two new palladium (II) complexes, [Pd(CAZ)(OH2 )2 ]2+ (1) and [Pd(3-AT)(OH2 )2 ]2+ (2), (CAZ=ceftazidime, and 3-AT=amitrole) were synthesized and studied for their potential as anticancer drugs with low toxicity and high potency. To fully characterize these complexes, we conducted elemental analysis and FT-IR studies. Furthermore, we irradiated the complexes with Indian 60 Co gamma rays and thoroughly evaluated their antimicrobial properties. Our results demonstrate that the inhibitory activity of complexes was significantly enhanced against (G+) bacteria and fungi. Additionally, we probed the complexes' interaction with CT-DNA and BSA using various techniques, including UV-vis spectroscopy, thermal denaturation, viscometry, gel electrophoresis, and molecular docking studies. Our findings conclusively demonstrate that these complexes possess a strong binding interaction with CT-DNA via minor groove binding and/or electrostatic interactions, as well as excellent binding affinity to BSA. Finally, we conducted a cytotoxicity assay that clearly indicates these complexes hold immense promise as cell growth inhibitors against MCF-7 and HCT-116.

Abstract 377: Developing a Novel Oxygen Carrier for Resuscitation and Oxygen Delivery Through Targeted Mutations of Recombinant Heme-Proteins

Introduction: Hemorrhage is a leading cause of death in trauma patients. Interventions dedicated to hemostatic resuscitation have demonstrated benefit in decreasing mortality due to hemorrhagic injury. There remain significant limitations to donor-based blood transfusions. We have identified a heme-binding protein, the Bacterial Regulator of carbon monoxide metabolism (RcoM), with suitable properties to be a robust artificial oxygen carrier. We have engineered a variant that lacks a DNA binding domain and contains cysteine mutations for improved stability (RcoM HBD-CCC). Hypothesis: We hypothesize that engineered RcoM HBD-CCC can be used as an ideal artificial oxygen carrier due to the optimization of 4 factors: ligand binding, nitrite reduction rate, autoxidation rate, and thermal denaturation. Methods: Spectrophotometric methods were used to characterize RcoM HBD-CCC in terms of its biochemical properties for optimal oxygen transport and release. Specifically, ligand binding was tested by determination of the P50 (oxygen pressure for 50% saturation of the molecule) value indicating the affinity of RcoM HBD-CCC towards oxygen. Nitrite reduction assays were carried out to test the rate at which nitric oxide (NO) forms to counteract potential NO scavenging. The autoxidation rate determines how long a protein stays in the reduced state, where it is able to bind to oxygen. Thermal denaturation (T m ) identifies the resistance of a protein to higher temperatures. Results: We determined that RcoM HBD-CCC’s P50 value was 4.2 mmHg which is within the ideal range for oxygen binding affinity given that optimal values for oxygen carriers to mimic hemoglobin oxygen dissociation are between 1 to 26 mmHg. Nitrite reductase rate was 5.6M -1 s -1 which is well above the rate for Hb (K = 0.12 M -1 s -1 ). RcoM HBD-CCC has a low autoxidation rate at 0.016 min -1 . RcoM HBD-CCC has a T m of 71°C which is well above all ambient environmental temperatures. Conclusion: We have designed and synthesized a variant of RcoM that we have demonstrated in in vitro spectrophotometric assays to have ideal biochemical properties for an artificial oxygen carrier. We next plan to test the efficacy of RcoM HBD-CCC in animal models of hemorrhage and assess safety in animal exposure models.

Variation of CI-2 Conformers upon Addition of Methanol to Water: An IMS-MS-Based Thermodynamic Analysis.

Chymotrypsin inhibitor 2 (CI-2) is a well-studied, textbook example of a cooperative, two-state, native ↔ denatured folding transition. A recent hybrid ion mobility spectrometry (IMS)/mass spectrometry (MS) thermal denaturation study of CI-2 (the well-studied truncated 64-residue model) in water reported evidence that this two-state transition involves numerous (∼41) unique native and non-native (denatured) solution conformations. The characterization of so many, often low-abundance, states is possible because of the very high dynamic range of IMS-MS measurements of ionic species that are produced upon electrospraying CI-2 solutions from a variable temperature electrospray ionization source. A thermodynamic analysis of these states revealed large changes in enthalpy (ΔH) and entropy (ΔS) at different temperatures, and it was suggested that such variation might arise because of temperature-dependent conformational changes of the protein in response to changes in the conformational entropy and the dielectric permeability of water, which drops from a value of ε ∼ 79 at 24 °C to ∼ 60 at 82 °C. Herein, we examine how adding methanol to water influences the distributions of CI-2 conformers and their ensuing stabilities. The dielectric constant of a 60:40 water:methanol (MeOH) drops from ε ∼ 60 at 24 °C to ∼ 51 at 64 °C. Although the same set of conformers observed in water appears to be present in 60:40 water:MeOH, the abundance of each is substantially altered by the presence of methanol. Relative free energy values (ΔG) and thermodynamic values [ΔH and ΔS and heat capacities (ΔCp)] are derived from a Gibbs-Helmholtz analysis. A comparison of these data from water and water:MeOH systems allows rare insight into how variations in solvation and temperature affect many-state protein equilibria. While these studies confirm that variations in solvent dielectric constant with temperature affect the distributions of conformers that are observed, our findings suggest that other solvent differences may also affect abundances.

Fate of pulse globulin proteins molecular Structure and composition on high moisture extrusion

Mungbean and lentil proteins are gaining attention for the production of high moisture extrusion (HME)-based meat- and dairy-analogues. However, understanding the mechanism of fibrous structure formation and molecular and higher-order changes in HME remains limited. Globulin protein isolates from mungbean (MBPI), green lentil (GLPI) and yellow pea (YPPI) along with commercial soy (CSPI) and yellow pea (CPPI) protein were investigated for their pasting properties using high-pressure rapid visco analyser (RVA), and the formation of anisotropic structures and molecular changes at constant HME conditions. Vicilin-rich MBPI showed higher viscosity on RVA, consumed higher specific mechanical energy (SME) during extrusion, and developed extrudates with higher textural strength and cutting force than proteins with both legumin and vicilin-like globulins, i.e. GLPI and YPPI. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography (SEC) of extrudates' soluble proteins showed dissociation of vicilin-like globulins into their smaller subunits. In contrast, legumin-like globulins were reaggregated into higher molecular weight soluble macromolecules. Further, SDS-PAGE of extrudates’ total protein revealed the involvement of legumin-like globulin in the macrostructure network of the extrudates. During HME, native unfolded structures were destroyed with conversion, predominately driving changes in the tertiary structure (thermal denaturation) and secondary structure with loss of helical structure to formation of random coil and beta-sheet structures. Protein extractability in different solvents was greatly reduced, with covalent and non-covalent interactions being majorly involved in the stabilisation of extrudates structure. Protein composition, and in particular vicilin-legumin ratio and protein conformation, determined the structural development and molecular changes in HME.

Exploring and strengthening the potential of R-phycocyanin from Nori flakes as a food colourant.

This study aimed to purify, characterise and stabilise the natural food colourant, R-phycocyanin (R-PC), from the red algae Porphyra spp. (Nori). We purified R-PC from dried Nori flakes with a high purity ratio (A618/A280≥3.4) in native form (α-helix content 53%). SAXS measurements revealed that R-PC is trimeric ((αβ)3) in solution. The thermal denaturation of α-helix revealed one transition (Tm at 52°C), while the pH stability study showed R-PC is stable in the pH range 4-8. The thermal treatment of R-PC at 60°C has detrimental and irreversible effects on R-PC colour and antioxidant capacity (22 % of residual capacity). However, immobilisation of R-PC within calcium alginate beads completely preserves R-PC colour and mainly retains its antioxidant ability (78 % of residual capacity). Results give new insights into the stability of R-PC and preservation of its purple colour and bioactivity by encapsulation in calcium alginate beads.

Mechanism of amino acid delay on thermal aggregation behavior of liquid egg yolk: Thermal aggregation, water distribution, molecular structure

Liquid Egg yolk (LEY) tend to aggregate during pasteurization and lead to pipe blockage, which has become a thorny problem in food industry. In this paper, the mechanism of two common amino acid osmolytes (betaine and proline) delaying thermal aggregation of LEY was investigated. The result showed that addition of 3% betaine or proline improved thermal denaturation temperature of LEY in rheology and also reduced its viscoelasticity. The addition of amino acid shrinked particle size distribution of LEY. Betaine LEY (BLEY) had strong surface hydrophobicity while proline LEY (PLEY) had excellent solubility, both of which maintained its emulsifying property. Scanning electron microscopy (SEM) revealed that BLEY and PLEY had more porous surfaces, and the structure was more loose compared with LEY at the same temperature. The relaxation time T2 indicated that betaine and proline can alleviate conversion of free water to immobilized and bound water during heat treatment process, thereby reducing the degree of thermal aggregation and viscosity. Fourier transform infrared spectroscopy (FT-IR) demonstrated the tendency of α-helix transformed to β-sheet as heating progress. Compared with LEY, the structure of BLEY and PLEY was more ordered, suggesting that orderliness of structure was the foundation for delaying thermal aggregation. In conclusion, this study provided a new idea and theoretical basis for production of heat-resistant LEY.

A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets invitro and signal-induced puncta invivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N-and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.

Mitochondriotropic agents conjugated with NSAIDs through metal ions against breast cancer cells

Two copper(I) polymorphs of formula [Cu(SALH)(TPP)3] (1a and 1b) were prepared by the conjugation of the Non-Steroidal Anti-Inflammatory Drug (NSAID) salicylic acid (SALH2) with the mitochondriotropic agent triphenylphosphine (TPP) via metal ion. For comparison, the isomorph [Ag(SALH)(TPP)3] (2) was prepared. The conjugates 1a, 1b and 2 were characterized by melting point (m.p.), Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, Ultraviolet-Visible (UV–Vis) spectroscopy and nuclear magnetic resonance (1H NMR). The crystal structures of 1a, 1b and 2 were confirmed by X-ray diffraction crystallography (XRD). The ex vivo binding affinity of 1–2 towards CT (calf thymus)-DNA was studied by UV, fluorescence, viscosity and DNA Thermal Denaturation studies. Their inhibitory activity against lipoxygenase (LOX) (an enzyme which is mainly located in the mitochondrion) was determined. The in vitro activity of 1–2 was evaluated against human breast cancer cell lines MCF-7 (hormone depended (HD)) and MDA-MB 281 (hormone independent (HI)) cells. Compounds 1–2 inhibit stronger than cisplatin the cancerous cells. The molecular mechanism of action of 1–2 was suspected by the MCF-7 cells morphology and confirmed by DNA fragmentation, Acridine Orange/Ethidium Bromide (AO/EB) Staining and mitochondrial membrane permeabilization tests.

Characterization of the thermal behavior and structural properties of a commercial high-solids confectionary gel made with gelatin

Gelatin plays a critical role in controlling the stability and texture of high-solids gels (HSGs), widely used in confectionery products and nutrient delivery systems. Most gelatin studies have been done on dilute solutions and films, rather than HSGs. Thus, the objectives of this study were to determine the: 1) thermal and structural characteristics of gelatin in a high-solids confectionary gel, 2) effect of renaturation temperature on reformation of gelatin helical structure, and 3) effect of time on loss and reformation of gelatin helical structure. Gelatin HSGs thermal and structural characteristics were investigated using differential scanning calorimetry (DSC), powder x-ray diffraction, and powder x-ray diffraction with total scattering pair distribution function analysis, respectively. The gelatin HSG DSC thermogram exhibited two thermal events: a glass transition and an endothermic peak associated with the helix-coil transition. Based on these results, the helix-coil transition was identified as a thermal denaturation event, not a melting event. The effect of renaturation temperature on gelatin reformation and the effect of time on the loss and reformation of triple-helix gelatin structure were determined using isothermal-DSC. Onset and peak denaturation temperatures increased with renaturation temperature, whereas the amount of triple-helix structure reformed increased with renaturation temperature up to 22 °C, then decreased. Both loss and renaturation of triple-helix structure were time-dependent processes. These results support the development of gelatin HSGs with controlled thermal stability and textural properties and contribute to solving the technical challenges associated with replacing gelatin in HSGs through enhanced understanding, obtained herein, of gelatin's thermal and structural characteristics.

Natural protein engineering in the Ω-loop: the role of Y221 in ceftazidime and ceftolozane resistance in Pseudomonas-derived cephalosporinase.

A wide variety of clinically observed single amino acid substitutions in the Ω-loop region have been associated with increased minimum inhibitory concentrations and resistance to ceftazidime (CAZ) and ceftolozane (TOL) in Pseudomonas-derived cephalosporinase and other class C β-lactamases. Herein, we demonstrate the naturally occurring tyrosine to histidine substitution of amino acid 221 (Y221H) in Pseudomonas-derived cephalosporinase (PDC) enables CAZ and TOL hydrolysis, leading to similar kinetic profiles (k cat = 2.3 ± 0.2 µM and 2.6 ± 0.1 µM, respectively). Mass spectrometry of PDC-3 establishes the formation of stable adducts consistent with the formation of an acyl enzyme complex, while spectra of E219K (a well-characterized, CAZ- and TOL-resistant comparator) and Y221H are consistent with more rapid turnover. Thermal denaturation experiments reveal decreased stability of the variants. Importantly, PDC-3, E219K, and Y221H are all inhibited by avibactam and the boronic acid transition state inhibitors (BATSIs) LP06 and S02030 with nanomolar IC50 values and the BATSIs stabilize all three enzymes. Crystal structures of PDC-3 and Y221H as apo enzymes and complexed with LP06 and S02030 (1.35-2.10 Å resolution) demonstrate ligand-induced conformational changes, including a significant shift in the position of the sidechain of residue 221 in Y221H (as predicted by enhanced sampling well-tempered metadynamics simulations) and extensive hydrogen bonding between the enzymes and BATSIs. The shift of residue 221 leads to the expansion of the active site pocket, and molecular docking suggests substrates orientate differently and make different intermolecular interactions in the enlarged active site compared to the wild-type enzyme.

Heterologous expression of Bacillus subtilis SL-44 glycosyltransferase catalyzed synthesis of ginsenoside Rh2

Abstract In this paper, the synthesis of ginsenoside Rh2 was catalyzed by using heterologous expression of Bacillus subtilis SL-44 glycosyltransferase. The synthesis parameters of ginsenoside Rh2 were optimized by the selection of strains and chemical supplies, the establishment of kinetic equations for the respiration rate of UGT enzyme, the effect of storage temperature on the model, and the glycosylation reaction of ginsenoside PPD with UGT. The effect of Rh2 saturation on the thermal denaturation temperature of the protein was analyzed along with the kinetic properties of the enzyme GE02773 (GE03484) while varying the saturation of Rh2. The results showed that the conversion of ginsenoside Rh2 reached 84% at a temperature of 35℃, pH 8, 5% DMSO, 0.4 of M-UDPG, and 1M-PPD in reaction with GE02773. In this paper, we successfully achieved the efficient synthesis of ginsenoside Rh2, which provides new ways and ideas for the application and synthesis of ginsenoside Rh2, with important practical significance and scientific value.

A single-domain protein catenane of dihydrofolate reductase.

A single-domain protein catenane refers to two mechanically interlocked polypeptide rings that fold synergistically into a compact and integrated structure, which is extremely rare in nature. Here, we report a single-domain protein catenane of dihydrofolate reductase (cat-DHFR). This design was achieved by rewiring the connectivity between secondary motifs to introduce artificial entanglement and synthesis was readily accomplished through a series of programmed and streamlined post-translational processing events in cells without any additional in vitro reactions. The target molecule contained few exogenous motifs and was thoroughly characterized using a combination of ultra-performance liquid chromatography-mass spectrometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protease cleavage experiments and ion mobility spectrometry-mass spectrometry. Compared with the linear control, cat-DHFR retained its catalytic capability and exhibited enhanced stability against thermal or chemical denaturation due to conformational restriction. These results suggest that linear proteins may be converted into their concatenated single-domain counterparts with almost identical chemical compositions, well-preserved functions and elevated stabilities, representing an entirely new horizon in protein science.

Experimental research on mechanism of collagen remodeling and excellent properties of skin incisions under dual beam laser welding

For problems of water loss caused by rapid energy dissipation and insufficient depth of heat-affected zone (HAZ) during skin welding with single laser beam. A dual beam 1064 nm semiconductor laser was used to weld incisions on the back of porcine skin, and the dual beam laser was consisted of preliminary beam and the secondary beam. The main beam is vertically incident, and the secondary beam is deflected by 30° on a vertical basis. In this study, there are three types of energy ratios of dual beam laser, which are 1:1, 2:1 and 3:1. From 77.22 J/cm2 to 135.4 J/cm2 stepped increasing energy density, the fusion performances of skin incisions after welding were comprehensively analyzed and characterized. Using Matlab and high-precision dynamometer to evaluate thermal denaturation and biomechanical properties, combined with H&E staining, Masson and Sirius red staining to analyze the microstructure characteristics, collagen deposition behavior and distribution around the HAZ of incisions after welding. The results showed that when the energy density was 102.96 J/cm2, at the energy ratio of 1:1, the collagen network structure was dense, collagen deposition was 48 %, thermal damage value was 0.39, and the immediate tensile strength was 206.3 N/cm2, which was far above the standard of wound healing performances after surgical treatment, and the thermal denaturation was controlled at a low level, realizing the reconstruction of collagen after fracture, achieving the optimal dual-beam laser welding performance, in vivo test results found that above laser type and energy ratio can reduce healing cycle into 7 days, which provided important references for later study on laser tissue welding technology.