Random Coil Content Research Articles

Conformational changes of proteins and oil molecules in fish oil/water interfaces of fish oil-in-water emulsions stabilized by bovine serum albumin

This study investigated the conformational changes in proteins and oil molecules at fish oil/water interfaces. Results of FT-IR indicate that α-helical content of bovine serum albumin decreased (from 46.1% to 28.4%), and β-sheet and random coil contents increased (from 15.5% and 13.5% to 18.9% and 28.1%, respectively) after protein adsorption at the fish oil/water interface. Fluorescence emission maximum of emulsions showed a blue shift compared to native proteins. FT-IR spectra of oil molecules showed broadenings in CH stretching vibration peaks of CH3 and CH2 groups of the fish oil. These results combined with structural information of BSA in the protein database led to the following hypothesis: binding occurs between proteins and lipid molecules in the oil-water interface. This study provides insight into the conformational changes of proteins and oil molecules in fish oil/water interfaces and broadens the fundamental understanding of the possible stability mechanisms of emulsions stabilized by proteins.

Effect of pH-shifting treatment on structural and functional properties of whey protein isolate and its interaction with (−)-epigallocatechin-3-gallate

Effect of pH-shifting on structural and functional properties of whey protein isolate and its interaction with (−)-epigallocatechin-3-gallate were investigated. Circular dichroism spectra showed that pH-shifting induced the decrease in α-helix content by 12.18% and β-sheet content by 3.24%, but β-turn and random coil content increased by 4.26% and 5.91%, respectively. Increase of fluorescence intensity and red-shift of maximum emission wavelength indicated the structural unfolding and exposure of tyrosine. The treatment also significantly increased the surface hydrophobicity, disulfide bonds content, solubility, emulsifying activity and emulsion stability of whey protein isolate at P < 0.05 level. Fluorescence quenching analysis revealed that treated whey protein isolate have a stronger binding affinity to (−)-epigallocatechin-3-gallate, resulting a better protection against the degradation of (−)-epigallocatechin-3-gallate and its antioxidant activity. This study confirmed that pH-shifting treatment can improve functional properties of whey protein isolate and its potential as a protective carrier for polyphones.

Characterization and functional evaluation of oat protein isolate-Pleurotus ostreatus β-glucan conjugates formed via Maillard reaction

Oat protein isolate is nutritious but with poor processing functionality. Pleurotus ostreatus β-glucan with good processing functionality can be conjugated with oat protein isolate via Maillard reaction, leading to an improved utilization of protein in food industry. Therefore, we produced conjugate with oat protein isolate and Pleurotus ostreatus β-glucan via Maillard reaction under controlled dry-heating conditions. The formation of conjugates with high molecular weight was identified by a new band of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analysis of amino acid composition showed that cysteine and lysine were the dominant Maillard reaction sites of oat protein isolate and Pleurotus ostreatus β-glucan. Changes in spatial configuration of conjugates caused reduction in their surface hydrophobicity of proteins and intrinsic fluorescence intensity. Fourier transform infrared spectroscopy analysis of conjugates suggested that Maillard reaction could cause the C=O stretching vibration, as well as the C-H and N-H deformation vibration. Circular dichroism analysis indicated that the secondary structure of conjugates was altered by decreasing the contents of α-helix and β-sheet and increasing the contents of β-turn and random coil. The surface structure of conjugates was loose and porous using scanning electron microscope. Furthermore, Maillard reaction could improve the solubility, emulsifying property and thermal stability of oat protein isolate. Our findings confirm the potential of protein-carbohydrate conjugate formed by Maillard reaction, to improve the application of instable but valuable proteins in food industry.

Effects of slit divergent ultrasound and enzymatic treatment on the structure and antioxidant activity of arrowhead protein

The arrowhead has attracted great research interest for their potential applications in pharmacy, food and biomedical areas. However, no information is reported about the nature and structure of the arrowhead protein (AP). Herein, effects of slit divergent ultrasound (28, 33, 40 KHz frequencies at 30–50 °C) and enzymatic (pepsin, trypsin, and alcalase) treatment on structure of AP were studied. In addition, changes in antioxidant activity of AP treated with ultrasound and enzymes were measured by chemical and cellular-based assays. The results showed that ultrasound treatment had considerable impact on the structure of AP and increased the susceptibility of AP to pepsin, trypsin and alcalase proteolysis. The changes in UV–Vis spectra, free sulfhydryl (SH) and disulfide bonds (SS) groups indicated that the structure of AP unfolded after ultrasound treatment. Besides, intrinsic fluorescence intensity of AP was increased by ultrasound treatment and then decreased after following enzymatic treatment. The circular dichroism (CD) analysis showed that ultrasound and enzymatic treatment decreased α-helix, β-turn of content of AP. However, the β-sheet and random coil content of AP increased. Interestingly, the AP after ultrasound and enzymatic treatment showed significant higher anti-oxidative activity in RAW 264.7 cells (p < 0.05) in comparison with control. In conclusion, the slit divergent ultrasonic provides a powerful endorsement for increasing the proteolysis of AP. Moreover, the improvement of the antioxidant activity of AP enzymatic hydrolysates provides a foundation of developing new type of plant-derived antioxidant peptides application.

Effects of Storage Time on Steamed Bread Quality and Gluten Protein Structure

The effects of storage time on the quality of steamed bread and the structure of gluten protein were investigated by determining the specific volume, the texture characteristics, the microstructure, the secondary structure of gluten and the moisture distribution. The results showed that with the increase of storage time, the specific volume of steamed bread gradually decreased. The hardness, stickiness and chewiness of steamed bread increased gradually, elasticity, cohesiveness and recovery decreased gradually. The contents of α-helix and β-turn in gliadin decreased, the contents of β-sheet and random coil increased. The contents of β-fold and random coils in glutenin increased, the contents of α-helix and β-turn gradually decreased. Both T21 and T22 decreased, the proportion of bound water gradually increased. The gluten protein and starch granules in steamed bread gradually break off, the gluten network also gradually collapsed and surface structure of steamed bread also changed from smooth to uneven.

Tuberculosis vaccine candidate: Characterization of H4-IC31 formulation and H4 antigen conformation

Tuberculosis (TB) is one of the leading causes of death worldwide, making the development of effective TB vaccines a global priority. A TB vaccine consisting of a recombinant fusion protein, H4, combined with a novel synthetic cationic adjuvant, IC31®, is currently being developed. The H4 fusion protein consists of two immunogenic mycobacterial antigens, Ag85 B and TB10.4, and the IC31® adjuvant is a mixture of KLK, a leucine-rich peptide (KLKL5KLK), and the oligodeoxynucleotide ODN1a, a TLR9 ligand. However, efficient and robust methods for assessing these formulated components are lacking. Here, we developed and optimized phase analysis light scattering (PALS), electrical sensing zone (ESZ), and Raman, FTIR, and CD spectroscopy methods to characterize the H4-IC31 vaccine formulation. PALS-measured conductivity and zeta potential values could differentiate between the similarly sized particles of IC31® adjuvant and the H4-IC31 vaccine candidate and could thereby serve as a control during vaccine formulation. In addition, zeta potential is indicative of the adjuvant to antigen ratio which is the key in the immunomodulatory response of the vaccine. ESZ was used as an orthogonal method to measure IC31® and H4-IC31 particle sizes. Raman, FTIR, and CD spectroscopy revealed structural changes in H4 protein and IC31® adjuvant, inducing an increase in both the β-sheet and random coil content as a result of adsorption. Furthermore, nanoDSF showed changes in the tertiary structure of H4 protein as a result of adjuvantation to IC31®. Our findings demonstrate the applicability of biophysical methods to characterize vaccine components in the final H4-IC31 drug product without the requirement for desorption.

Structural characterization of recombinant streptokinase following recovery from inclusion bodies using different chemical solubilization treatments

Circular dichroism (CD) in far-UV region was employed to study the extent of changes occurred in the secondary structures of recombinant streptokinase (rSK), solubilized from inclusion bodies (IBs) by different chemicals and refolded/purified by chromatographic techniques. The secondary structure distribution of rSK, obtained following different chemical solubilization systems, was varied and values in the range of 12.4–14.5% α-helices, 40–51% β-sheets and 35.5–48.3% turns plus residual structures were found. With reducing the concentration of chemicals during IB solubilization, the content of turns plus random coils was diminished and simultaneously the amounts of α- and β-sheets were increased. These changes in the secondary structures would lower the hydrophobicity along with the chance of protein aggregation and expose the hydrophilic regions of the protein. Therefore, these alterations in the secondary structures, occurred following efficient IBs solubilization by low concentration of chemicals, could be related to enhancement in rSK biological potency previously observed.

Effects of High-Pressure Homogenization at Different Pressures on Structure and Functional Properties of Oyster Protein Isolates

Abstract Oyster protein isolate (OPI) suspensions (6.19 % ± 0.82 %, w/v) were treated by high-pressure homogenization (HPH) at 0 (control), 20, 40, 60, 80 or 100 MPa for three cycles. Protein profiles, secondary structure, free sulfhydryl, surface hydrophobicity, particle size distribution, zeta-potential, solubility, water and oil holding capacity (OHC), emulsifying and foaming properties of the obtained suspensions were analyzed. The results showed that HPH treatment did not cause changes in protein profiles of OPI, but caused changes in secondary structure, content of α-helix decreased but content of β-turn and random coil increased significantly (P &lt; 0.05). Free sulfhydryl and surface hydrophobicity all increased significantly (P &lt; 0.05) after HPH treatment, indicating that tertiary and quaternary structures changed. Functional properties of OPI significantly (P &lt; 0.05) improved after HPH treatment, such as zeta-potential (from −12.67 to −33.57 mV), solubility (from 20.24 % to 57.99 %), OHC (from 981.77 % to 1229.40 %), foaming ability (from 17.50 % to 35.00 %), foaming stability (from 44.49 % to 66.60 %), emulsifying activity index (from 8.87 to 17.06 m2/g) and emulsion stability index (from 14.65 to 41.68 min). At 60 MPa and 80 MPa, the improvements were more remarkable. However, HPH treatment significantly (P &lt; 0.05) decreased particle size (from 200–500 nm to 0–200 nm) and water holding capacity (from 341.15 % to 216.96 %). These improvements were closely related to structural changes and reduction of particle size. Application of different pressures affected functional properties of OPI. These results could provide information for determining HPH applying condition in OPI modification.

The effect of ultrasound on the properties and conformation of glucoamylase

In this study, the effects of ultrasonic treatment on the enzyme activity of glucoamylase were investigated and the mechanism of the effect was explored by determining their conformational changes. The enzyme activity was increased by 21.07% over the control with ultrasound at 420W for 10min at 60°C, and Vmax and Km values of glucoamylase were also increased. After ultrasonic treatment at 420W, thermodynamic experiments of glucoamylase showed that Ea, ΔG, ΔH decreased but slight increase in ΔS was observed. In addition, after the treatment, the UV spectra showed that the number of tryptophan and tyrosine on glucoamylase surface increased, and the contents of α-helix and random coil increased by 17.8%, 12.41%, respectively, through the circular dichroism spectrum scanning. Besides, fluorescence spectra indicated that the absorption of tryptophan has a red shift. Furthermore, fourier transform infrared spectra suggested that the glucoamylase was significantly influenced by ultrasound. The results indicated that the activity of glucoamylase could be modified by ultrasonic treatment mainly in terms of the variation of their conformation.

Effects of sonication on the physicochemical and functional properties of walnut protein isolate

The objective of this study was to investigate the impact of high-intensity ultrasound treatment (sonication) on the molecular, physicochemical, and functional properties of walnut protein isolate. Aqueous walnut protein suspensions were sonicated at varying power levels (200, 400 or 600 W) and times (15 or 30 min), and then any alternations in protein structure and properties were determined. SDS-PAGE demonstrated that there were no changes in protein electrophoretic patterns, indicating that sonication did not break covalent bonds. Circular dichroism spectroscopy indicated a small change in protein secondary structure after sonication, with a decrease in α-helix and increase in β-sheet, β-turn, and random coil content. There was an increase in surface free sulfhydryl (SH) groups and a decrease in fluorescence intensity after sonication, indicating that appreciable changes in tertiary structure occurred. Ultrasound reduced the size of the particles in aqueous walnut protein dispersions as confirmed by static light scattering and scanning electron microscopy, suggesting that sonication dissociated protein aggregates. Moreover, the water-solubility (+22%), emulsifying activity index (+26%), and emulsifying stability index (+41%) all increased after sonication. These results suggest that sonication is a valuable tool for improving the functional attributes of walnut proteins.

Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory Factor as a Molecular Redox Switch.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased β-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

Effect of glucose glycosylation following limited enzymatic hydrolysis on functional and conformational properties of black bean protein isolate

The effect of glycosylation following limited enzymatic hydrolysis on conformational and functional properties of black bean protein isolate (BBPI) was investigated in this study. The black bean protein hydrolysate (HBBPI) was prepared using alcalase at pH 9.0 and then glycosylated with glucose (G) at 80 °C for different incubation times from 1 to 6 h. Grafted HBBPI with glucose (HBBPI-G conjugates) had a higher molecular weight than HBBPI by SDS-PAGE analysis. HBBPI-G conjugates had a higher content of β-turn and random coil, but a lower content of α-helix and β-sheet structure than BBPI. HBBPI-G conjugates had lower fluorescence intensity and exhibited bathochromic shift compared with BBPI. Subsequently, the functional properties were also evaluated. Results indicated that the emulsifying activity and solubility were obviously improved (P < 0.05) by HBBPI-G conjugates incubated for 4 h with 6.5% degree of hydrolysis compared to BBPI. Additionally, the glycosylation had positive effects on the reducing power and hydroxyl radical scavenging rate. Therefore, the combination of limited hydrolysis and glycation can be used as an effective method for BBPI modification to obtain enhanced functional properties.

Biosurfactant-Protein Interaction: Influences of Mannosylerythritol Lipids-A on β-Glucosidase.

In this work, the influences of a biosurfactant, mannosylerythritol lipids-A (MEL-A) toward β-glucosidase activity and their molecular interactions were studied by using differential scanning calorimetry (DSC), circular dichroism spectroscopy (CD), isothermal titration calorimetry (ITC), and docking simulation. The enzyme inhibition kinetics data showed that MEL-A at a low concentration (< critical micelle concentration (CMC), 20.0 ± 5.0 μM) enhanced β-glucosidase activity, whereas it inhibited the enzyme activity at higher concentrations more than 20.0 μM, followed by a decreased Vmax and Km of β-glucosidase. The thermodynamics and structural data demonstrated that the midpoint temperature (Tm) and unfolding enthalpy (ΔH) of β-glucosidase was shifted to high values (76.6 °C, 126.3 J/g) in the presence of MEL-A, and the secondary structure changes of β-glucosidase, including the increased α-helix, β-turn, or random coil contents, and a decreased β-sheet content were caused by MEL-A at a CMC concentration. The further ITC and docking simulations suggested the bindings of MEL-A toward β-glucosidase were driven by weak hydrophobic interactions happened between the amino acid residues of β-glucosidase and the fatty acid residues of MEL-A, in addition to hydrogen bonds between amino acids and hydroxyl in glycosyl residues of this biosurfactant.

Swirling cavitation improves the emulsifying properties of commercial soy protein isolate

Since emulsifying properties are important functional properties of soy protein, many physical, chemical, and enzymatic methods have been applied to treat soy protein to improve emulsifying properties. In this study, we investigated the effects of swirling cavitation at different pressures and for different times on emulsifying and physicochemical properties of soy protein isolate (SPI). The SPI treated with swirling cavitation showed a significant decrease in particle size and increase in solubility. Emulsions formed from treated SPI had higher emulsifying activity and emulsifying stability indexes, smaller oil droplet sizes, lower flocculation indexes, higher adsorbed proteins, lower interfacial protein concentrations, and lower creaming indexes than those formed from untreated SPI, indicating that swirling cavitation improved the emulsifying properties of the SPI. Furthermore, swirling cavitation treatment significantly enhanced the surface hydrophobicity, altered the disulfide bond and exposed sulfhydryl group contents of the SPI. The secondary structure of the SPI was also influenced by swirling cavitation, with an increase in β-sheet content and a decrease in α-helix, β-turn, and random coil contents. In addition, several significant correlations between physicochemical and emulsifying properties were revealed by Pearson correlation analysis, suggesting that the physicochemical changes observed in treated SPI, including the decreased particle size, increased solubility and surface hydrophobicity, and enhanced β-sheet formation, may explain the improved emulsifying properties of the isolate. Thus, our findings implied that swirling cavitation treatment may be an effective technique to improve the emulsifying properties of SPI.

The functional properties and structural characteristics of deamidated and succinylated wheat gluten

Wheat gluten protein (WGP) was modified by deamidation and succinylation to improve its functional properties. The properties of deamidated and succinylated WGP were compared with those of unmodified WGP, deamidated WGP and succinylated WGP. Structural analysis was carried out with FTIR. Surface visualization was carried out with SEM and the thermal stability was determined by DSC. The results showed that an acylation level of 73.64% produced the optimal properties for modified WGP. After modification, its functional properties were significantly improved. Modification did not significantly change the α-helical content, but there was a significant decrease in its β-turn content and significant increases in β-sheet and random coil content. According to SEM images, the modified WGP exhibited a smooth and evenly distributed sheet-like structure. The thermal stability of WGP was slightly decreased by modification. These results provide important information for the use of modified WGP in novel products. For example, adding deamidated and succinylated WGP to pound cake may reduce the amount of butter needed by up to 20%.

Effects of Dielectric Barrier Discharge (DBD) Cold Plasma Treatment on Physicochemical and Functional Properties of Peanut Protein

Peanut protein isolate (PPI) solutions were modified by dielectric barrier discharge (DBD) cold plasma (CP) treatment. Effects of CP treatment on the solubility, emulsion stability, and water holding capacity (WHC) of peanut protein were studied. The results showed a significant improvement in solubility, emulsion stability, and WHC following CP treatment. CP treatment resulted in the unfolding of PPI structure, thereby increasing the β-sheet and random coil content and decreasing the α-helix and β-turn content, as analyzed by Fourier-transform infrared spectroscopy. Low-field nuclear magnetic resonance showed an increase in the peak area of T 21 relaxation time by CP treatment and the change in T 21 peak area was in agreement with the result of WHC. This study demonstrated that CP may be successfully applied as a method to modify the functionality of PPI.

Mechanism of Reduction in IgG and IgE Binding of β-Lactoglobulin Induced by Ultrasound Pretreatment Combined with Dry-State Glycation: A Study Using Conventional Spectrometry and High-Resolution Mass Spectrometry.

Bovine β-lactoglobulin (β-Lg) is one of major allergens in cow's milk. Previous study showed that ultrasound treatment induced the conformational changes of β-Lg and promoted the glycation in aqueous solutions, which is, however, less efficient compared with dry-state. In this work, the effect of ultrasound pretreatment combined with dry-state glycation on the IgG and IgE binding of β-Lg was studied. Dry-state glycation with mannose after ultrasound pretreatment at 0-600 W significantly reduced the IgG and IgE binding of β-Lg, with the lowest values observed at 400 W. The decrease in the IgG and IgE binding of β-Lg was attributed to the increase in glycation extent and the changes of secondary and tertiary structure, which reflected in the increase of UV absorbance, α-helix and β-sheet contents, as well as the decrease of intrinsic fluorescence intensity, surface hydrophobicity, β-turn, and random coil contents. Moreover, ultrasound pretreatment promoted the reduction of IgG and IgE binding abilities by improving glycation, reflecting in the increase of the glycation sites and the degree of substitution per peptide (DSP) value determined by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Ultrasound pretreatment at 400 W showed the most significantly enhanced glycation extent. Besides, the results suggested FTICR-MS could provide insights into the glycation at molecular level, which was conducive to the understanding of the mechanism of the reduction in the IgG and IgE binding of β-Lg. Therefore, ultrasound pretreatment combined with dry-state glycation may be a promising method for β-Lg hyposensitization.

Molecular dynamics simulation of the interaction between dense-phase carbon dioxide and the myosin heavy chain

Dense-phase carbon dioxide (DPCD) can induce myosin denaturation and aggregation and result in the formation of a gel. To explore the mechanism that DPCD induce myosin the formation of gels, molecular dynamics simulations were used to investigate the effects of DPCD on the structural properties of the myosin heavy chain (MHC, GenBank Accession No: BAM65721.1) from Litopenaeus vannamei. In addition, the interaction between the MHC and CO2 was explored under the coupling of pressure and temperature. To facilitate a better understanding and comparison, the structure of MHC was simulated in water at 0.1MPa and 50°C (called the myosin-water system, MWS) and in water and DPCD at 25MPa and 50°C (called the myosin-water-DPCD system, MWDS). The analysis of the root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) revealed that the conformation of the MHC in both MWDS and MWS changed significantly relative to its initial structure. Moreover, the results of the solvent-accessible surface area demonstrated that the MHC structure changes from a compact arrangement to a looser arrangement in both MWDS and MWS. Finally, the results from investigations into the secondary structures showed that the α-helix content of the MHC decreased, whereas the β-turn and random coil content increased. Greater overall structural variations of the MHC were observed in MWDS than those observed in MWS. DPCD exerts a substantial effect on the MHC structure because of its hydrogen bonding, electrostatic repulsion and hydrophobic interactions with the MHC.

Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide.

Previously, we have shown that serine-16 phosphorylation in native full-length porcine amelogenin (P173) and the Leucine-Rich Amelogenin Peptide (LRAP(+P)), an alternative amelogenin splice product, affects protein assembly and mineralization in vitro. Notably, P173 and LRAP(+P) stabilize amorphous calcium phosphate (ACP) and inhibit hydroxyapatite (HA) formation, while non-phosphorylated counterparts (rP172, LRAP(−P)) guide the growth of ordered bundles of HA crystals. Based on these findings, we hypothesize that the phosphorylation of full-length amelogenin and LRAP induces conformational changes that critically affect its capacity to interact with forming calcium phosphate mineral phases. To test this hypothesis, we have utilized Fourier transform infrared spectroscopy (FTIR) to determine the secondary structure of LRAP(−P) and LRAP(+P) in the absence/presence of calcium and selected mineral phases relevant to amelogenesis; i.e., hydroxyapatite (HA: an enamel crystal prototype) and (ACP: an enamel crystal precursor phase). Aqueous solutions of LRAP(−P) or LRAP(+P) were prepared with or without 7.5 mM of CaCl2 at pH 7.4. FTIR spectra of each solution were obtained using attenuated total reflectance, and amide-I peaks were analyzed to provide secondary structure information. Secondary structures of LRAP(+P) and LRAP(−P) were similarly assessed following incubation with suspensions of HA and pyrophosphate-stabilized ACP. Amide I spectra of LRAP(−P) and LRAP(+P) were found to be distinct from each other in all cases. Spectra analyses showed that LRAP(−P) is comprised mostly of random coil and β-sheet, while LRAP(+P) exhibits more β-sheet and α-helix with little random coil. With added Ca, the random coil content increased in LRAP(−P), while LRAP(+P) exhibited a decrease in α-helix components. Incubation of LRAP(−P) with HA or ACP resulted in comparable increases in β-sheet structure. Notably, however, LRAP(+P) secondary structure was more affected by ACP, primarily showing an increase in β-sheet structure, compared to that observed with added HA. These collective findings indicate that phosphorylation induces unique secondary structural changes that may enhance the functional capacity of native phosphorylated amelogenins like LRAP to stabilize an ACP precursor phase during early stages of enamel mineral formation.

Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase from Coprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity.

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Δ5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Δ5C contained less α-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45°C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Δ5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Δ5C exhibited a much lower Km value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Δ5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Δ5C a good model for the structure-function study of (α/β)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.