Changes In Tertiary Structure Research Articles

Amino acids as stabilizers for lysozyme during the spray-drying process and storage

Amino acids (AAs) have been used as excipients in protein formulations both in solid and liquid state products due to their stabilizing effect. However, the mechanisms by which they can stabilize a protein have not been fully elucidated yet. The purpose of this study was to investigate the effect of AAs with distinct physicochemical properties on the stability of a model protein (lysozyme, LZM) during the spray-drying process and subsequent storage. Molecular descriptor based multivariate data analysis was used to select distinct AAs from the group of 20 natural AAs. Then, LZM and the five selected AAs (1:1 wt ratio) were spray-dried (SD). The solid form, residual moisture content (RMC), hygroscopicity, morphology, secondary/tertiary structure and enzymatic activity of LZM were evaluated before and after storage under 40 °C/75 % RH for 30 days. Arginine (Arg), leucine (Leu), glycine (Gly), tryptophan (Trp), aspartic acid (Asp) were selected because of their distinct properties by using principal component analysis (PCA). The SD LZM powders containing Arg, Trp, or Asp were amorphous, while SD LZM powders containing Leu or Gly were crystalline. Recrystallization of Arg, Trp, Asp and polymorph transition of Gly were observed after the storage under accelerated conditions. The morphologies of the SD particles vary upon the different AAs formulated with LZM, implying different drying kinetics of the five model systems. A tertiary structural change of LZM was observed in the SD powder containing Arg, while a decrease in the enzymatic activity of LZM was observed in the powders containing Arg or Asp after the storage. This can be attributed to the extremely basic and acidic conditions that Arg and Asp create, respectively. This study suggests that when AAs are used as stabilizers instead of traditional disaccharides, not only do classic vitrification theory and water replacement theory play a role, but the microenvironmental pH conditions created by basic or acidic AAs in the starting solution or during the storage of solid matter are also crucial for the stability of SD protein products.

Probing Enzymatic PET Degradation: Molecular Dynamics Analysis of Cutinase Adsorption and Stability.

Understanding the mechanisms influencing poly(ethylene terephthalate) (PET) biodegradation is crucial for developing innovative strategies to accelerate the breakdown of this persistent plastic. In this study, we employed all-atom molecular dynamics simulation to investigate the adsorption process of the LCC-ICCG cutinase enzyme onto the PET surface. Our results revealed that hydrophobic, π-π, and H bond interactions, specifically involving aliphatic, aromatic, and polar uncharged amino acids, were the primary driving forces for the adsorption of the cutinase enzyme onto PET. Additionally, we observed a negligible change in the enzyme's tertiary structure during the interaction with PET (RMSD = 1.35 Å), while its secondary structures remained remarkably stable. Quantitative analysis further demonstrated that there is about a 24% decrease in the number of enzyme-water hydrogen bonds upon adsorption onto the PET surface. The significance of this study lies in unraveling the molecular intricacies of the adsorption process, providing valuable insights into the initial steps of enzymatic PET degradation.

Molecular epidemiological investigation and recombination analysis of Cachavirus prevalent in China.

Chaphamaparvovirus carnivoran1 (canine Chaphamaparvovirus, also known as Cachavirus [CachaV]) is a novel parvovirus first reported in dog feces collected from the United States in 2017 and China in 2019. To continuously track its infection and evolution status, 276 canine anal swabs were obtained from pet hospitals in central, northern, and eastern China between 2021 and 2023 and screened via polymerase chain reaction; subsequently, a systematic study was conducted. Of these samples, nine (3.3%) were positive for CachaV. Using polymerase chain reaction, whole genome sequences of the nine CachaV-positive strains were amplified. The NS1 amino acid sequence identity between CachaV strains from China and other countries was 96.23-99.85%, whereas the VP1 protein sequence identity was 95.83-100%. CHN230521 demonstrated the highest identity for NS1 amino acids (99.85%) and VP1 amino acids (100%) with NWT-W88 and CP-T015. According to the model prediction of CHN220916-VP1 protein, Met64Thr, Thr107Ala, and Phe131Ser mutations may cause tertiary structural changes in VP1 protein. Interestingly, each of the nine CachaV strains harbored the same site mutations in NS1 (Ser252Cys, Gly253Leu, and Gly254Thr). Although no explicit recombination events were predicted, the clustering and branching of the phylogenetic tree were complicated. Based on the evolution trees for VP1 and NS1, the nine CachaV strains identified from 2021 to 2023 were closely related to those identified in gray wolves and cats. This study may be beneficial for evaluating the prevalence of CachaVs in China, thereby understanding the evolution trend of CachaVs.

Studies on the effects of preheated β-lactoglobulin on the physicochemical properties of theaflavin-3,3′-digallate and the interaction mechanism

Teaflavin-3,3′-digallate (TFDG) is a sensory and health benefit substance in tea beverage, but is usually eliminated during processing due to the formation of tea cream by binding with proteins. In order to retain the TFDG, β-lactoglobulin (β-LG) was used to bind with TFDG and avoid the formation of tea cream. The results of physicochemical tests and entropy weight-TOPSIS showed that the best β-LG-TFDG dispersion (TFDG concentration: 0.468 g/L) had low particle size (37.29 nm), high electrical potential (−32.43 mv), high transmittance (88.52%), and strong antioxidant activity (84.82%), when the preheated temperature of β-LG was 85 °C and the molar ratio was 1:2. The multispectral experiment results showed that TFDG interacted with β-LG via static quenching, and preheated treatment of β-LG enhanced the interaction between β-LG and TFDG. CD spectroscopy suggested that TFDG induced slight changes in the secondary structure of β-LG. The results of molecular dynamics showed that preheated treatment leading in the changes in tertiary structure of β-LG, opening of key ring region of β-LG and exposing the active cavities of β-LG, which enhances the intermolecular forces between TFDG and β-LG. What's more, molecular docking was proved that preheated treatment of β-LG enhances the hydrophobic interactions and hydrogen bonds, which dominated the interaction between TFDG and β-LG. The results proposed in this research could further reducing tea cream formation and improving the healthy and sensory performances of tea beverage by using preheated β-LG without preserving both the tea pigments and the active nutrients.

Biophysical insight into the binding mechanism of epigallocatechin-3-gallate and cholecalciferol to albumin and its preventive effect against AGEs formation: An in vitro and in silico approach

Advanced glycation end products (AGEs) are produced non-enzymatically through the process of glycation. Increased AGEs production has been linked to several diseases including polycystic ovary syndrome (PCOS). PCOS contributes to the development of secondary comorbidities, such as diabetes, cardiovascular complications, infertility, etc. Consequently, research is going on AGEs-inhibiting phytochemicals for their potential to remediate and impede the progression of hyperglycaemia associated disorders. In this study human serum albumin is used as a model protein, as albumin is predominantly present in follicular fluid. This article focusses on the interaction and antiglycating potential of (−)-Epigallocatechin-3-gallate (EGCG) and vitamin D in combination using various techniques. The formation of the HSA-EGCG and HSA-vitamin D complex was confirmed by UV and fluorescence spectroscopy. Thermodynamic analysis verified the spontaneity of reaction, and presence of hydrogen bonds and van der Waals interactions. FRET confirms high possibility of energy transfer. Cumulative antiglycation resulted in almost 60 % prevention in AGEs formation, decreased alterations at lysine and arginine, and reduced protein carbonylation. Secondary and tertiary structural changes were analysed by circular dichroism, Raman spectroscopy and ANS binding assay. Type and size of aggregates were confirmed by Rayleigh and dynamic light scattering, ThT fluorescence, SEM and SDS-PAGE. Effect on cellular redox status, DNA integrity and cytotoxicity was analysed in lymphocytes using dichlorofluorescein (DCFH-DA), DAPI and MTT assay which depicted an enhancement in antioxidant level by cumulative treatment. These findings indicate that EGCG and vitamin D binds strongly to HSA and have antiglycation ability which enhances upon synergism.

Abstract 6344: Engineering of ICAM-1 refractory antibodies for the development of therapeutic antibodies and antibody drug conjugates (ADCs) in ICAM-1 over-expressing cancers

Abstract Human cancers employ a number of mechanisms to evade host immune responses against novel antigens generated from aberrant over-expression, mutations and/or epigenetic alterations. Humoral immunity utilizes antibodies and immune-effector cells as well as molecular immune complexes involving the complement system to mediate the killing of dysregulated cancer cells. We refer to these anti-cancer mechanisms as Humoral Immuno-Oncology (HIO). HIO suppression is mediated by tumor-produced proteins called HIO factors. One such factor is CA125, which was previously shown to bind IgG-type antibodies and inhibit their immune-effector activities, including antibody dependent (ADCC) and complement dependent (CDC) cellular cytotoxicity. Using a combination of experimental screening and literature searches, a second protein produced by tumors and associated with a variety of cancer indications was discovered and found to be soluble ICAM-1 (sICAM-1). Through various functional studies we reported that sICAM-1 and membrane ICAM-1 (mICAM-1) are capable of binding IgG1-type antibodies that in turn inhibits their immune-effector activity. Through a combination of truncation and substitution mutagenesis, we identified a four amino acid motif within the CH3 domain of IgG1 essential for ICAM-1 binding, resulting in the inhibition of ADCC activity. While traditional glycine and alanine substitutions in this region abrogated ICAM-1 binding, these modifications caused tertiary structural changes in the Fc domain that resulted in loss of ADCC activity. Through a combinatorial amino acid substitution approach, we identified a four amino acid combination within the 407YSKL410 to 407FARV410 motif that resulted in antibodies refractory to sICAM-1 binding with robust ADCC activity. Additionally, isogenic wildtype and ICAM-1 knockdown target cell lines showed mICAM-1 inhibited target cell killing of a saporin antibody drug conjugate (ADC) and this was the direct result of mICAM-1 reducing antibody internalization, a requisite for maximal ADC target cell killing. The inhibitory activity of mICAM-1 could be overcome employing antibodies containing the 407FARV410 substitution. These findings highlight yet another mechanism by which tumors can suppress the host’s immune system for survival and offer new concepts for developing antibody-based therapies that can aid in the treatment of various cancer indications, especially those over-expressing ICAM-1. Moreover, the findings here offer diagnostic and therapeutic clinical design opportunities to improve upon existing approved immune-mediated therapies for which this factor is present. Citation Format: Brad Kline, Luigi Grasso, Nicolas Nicolaides. Engineering of ICAM-1 refractory antibodies for the development of therapeutic antibodies and antibody drug conjugates (ADCs) in ICAM-1 over-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6344.

Abstract 4890: In silico study of enzymes involved in heparan sulfate catabolic process in cancer resistant mammals

Abstract Some mammals such as elephants, bats, whales, and blind mole rats (Spalax) are resistant to cancer. One of the cancer protective mechanisms present in such species includes a reduced degradation of heparan sulfate (HS), an important component of the extracellular matrix (ECM), present in Spalax. Such effect occurs due to a splicing variant of heparanase, called variant 36, that lacks enzymatic activity. Consequently, both HS and ECM are very stable in Spalax’s tissues. If HS is stabilized, in cancer resistant mammals, due to the decrease of heparanase or other enzymes involved in HS catabolic process is unknown. We studied total of 9 enzymes (HPSE2, GusB, IDUA, IDS, NAGLU, SGSH, GNS, Sulf-2, HGSNAT) that regulate HS catabolism and homologs in 9 mammals. Multiple sequence alignments were done by MEGA to identify substitutions present in cancer resistant species in comparison with cancer susceptible animals. Such amino acid variants were further analyzed and both their energy stability and their functional effects were predicted by using online in silico tools including MAESTROweb, Condel, PANTHER, REVEL, and CADD. Finally, homologs with deleterious predicted variants were modelled with SWISSMODEL and their 3D structures were analyzed with Pymol and ChimeraX to identify structural features likely related to its loss of function. We found two loss of function missense variants, K264Q and W626L, in Alpha-L-iduronidase (IDUA) of Spalax. In addition, most of Brandt's bat enzymes and the N-sulphoglucosamine sulphohydrolase (SGSH) of the bowhead whale were very energy destabilizing enzymes with positive and higher ΔΔG values compared to human homologs. In the other hand, although some catalytic residues in the study enzymes are conserved in cancer resistant animals, their spatial arrangement and conformation in the 3D structure are different compared to human homologs, including E182 of IDUA which is one of the catalytic residues. In general, the secondary structure is conserved across the 9 enzymes studied but some differences were observed in cancer resistant animals including one of the beta strands near the catalytic domain in human and mice IDUA that appears as a loop in Spalax. In summary, HS stabilization could raise in cancer resistant mammals through the existence of lack of enzymatic activity not only in heparanase but also in other enzymes that participate in HS catabolic process, via a set of loss of function variants and changes in secondary and tertiary structure as well as a reduction of energy stabilization. More interestingly, such mechanism could be present in other cancer resistant mammals such as bats and bowhead whales. This study demonstrates that computational approaches are useful to study biological mechanisms of cancer resistance present in some mammals. This knowledge could be translated to humans and HS catabolic process could offer a new source of therapeutic anticancer targets. Citation Format: Fabrizio Oliveri Payet, Claudia Machicado Rivero. In silico study of enzymes involved in heparan sulfate catabolic process in cancer resistant mammals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4890.

5-methyl-salicylaldoxime based ionic liquids for non-destructive separation of protein and cadmium

Confronting with explosive cadmium contaminations to environment, non-destructive separation of protein with cadmium for protecting food safety is hardpressed. Ionic liquids (ILs) are benign bio-compatible stabilizer of proteins with considerable metal affinity, while their application in separation of cadmium from protein is limited owing to their comparative metal affinities with protein. Accordingly, we firstly synthesize 1-methyl-3 (salicylaldoxime-5-methyl) imidazole hexafluorophosphate ([Salenmim][PF6]) to enhance cadmium extraction with hydroxyl and oxime groups as ligands of cadmium. Structure of [Salenmim][PF6] is confirmed by 1H NMR, 13C NMR, 31P NMR, 19F NMR, FT-IR, Raman spectra, and theoretical simulations. [Salenmim][PF6] can remove 85.31 % of Cd2+ from bovine serum albumin (BSA) at pH of 2.0. X-ray photoelectron spectroscopy (XPS) of BSA and [Salenmim][PF6] before and after Cd2+ extraction illustrate that Cd2+ generally bonds with C = N and O-H of [Salenmim][PF6] to achieve detachment of Cd2+ from BSA. Molecular docking of BSA and Cd2+ indicates that 4 amino acids residues listed as ASP561, ASP562, LYS563, and GLU564 coordinate with Cd2+ with coordinate number close to 1, further confirmed as 1.48 by isothermal titration calorimetry (ITC) analysis. DFT calculations of [Salenmim][PF6] with Cd2+ demonstrate that energy gap between HOMO orbital localized at benzene ring is 1.6860 eV higher than LUMO orbital, faciliating formation of [Salenmim][PF6]-Cd2+ complex. Finally, there are no changes in secondary and tertiary structure of BSA and no ILs residual observed after [Salenmim][PF6] assisted Cd2+ extraction. This work firstly reports a method for synthesis of [Salenmim][PF6] with high affinity to cadmium and systemically reveals how do structure of [Salenmim][PF6] enhance heavy metal removal from proteins, providing a possible strategy for heavy metal extraction from proteins.

Insight into the mechanisms of combining direct current magnetic field with phosphate in promoting emulsifying properties of myofibrillar protein

This study investigated the combined effects of direct-current magnetic field (DC-MF, 9.5 mT) and tetrasodium-pyrophosphate (TSPP, 1-5 g/L) on emulsified gel properties of porcine myofibrillar protein (MP). Results showed that MP at DC-MF and 3 g/L TSPP had decreased spectrum intensity of UV and fluorescence compared to that without DC-MF, owing to the changes of MP tertiary structure caused by DC-MF, especially tryptophan and tyrosine. The emulsion treated with DC-MF behaved better emulsifying activity and stability than that without DC-MF under such condition. And emulsion had lower creaming index and better storage stability. Gels prepared by this MP emulsion had low porosity and stable structure, accompanying with smaller size and more uniform distribution of oil droplets. Microstructure images showed that gels were covered with microporous structure, which was conducive to the good WHC of the emulsified gels (97.12%). These results showed the feasibility of DC-MF and TSPP in improving MP emulsion/emulsified gel.

Preparation of housefly (Musca domestica) larvae protein hydrolysates: Influence of dual-sweeping-frequency ultrasound-assisted enzymatic hydrolysis on yield, antioxidative activity, functional and structural attributes

Dual-sweeping-frequency ultrasound (DSFU) was utilized in the preparation of polypeptides from housefly (Musca domestica) larvae protein (HLP). Results indicated that ultrasonication (20 ± 2/28 ± 2 kHz, 42 W/L, 25 min) significantly increased peptide yield and DPPH scavenging capacity by 8.25 % and 14.83 %, respectively. Solubility, foaming and emulsification properties of polypeptides were improved by 19.89 %, 33.33 % and 38.74 % over the control; along with notable reduction in particle size and increase in zeta potential. Tertiary structural changes of the sonicated hydrolysates were illustrated by UV and fluorescence spectra. FTIR showed that ultrasonication increased α-helix, β-turn, and random coil by 38.23 %, 46.35 % and 16.36 %, respectively, but decreased β-sheet by 48.03 %, indicating partial unfolding in HLP hydrolysate conformation and reduction in intermolecular interactions. The research results demonstrated that dual-sweeping-frequency ultrasonication has a great prospect in industry application for the purpose of improving enzymolysis efficiency and product quality for housefly larvae protein hydrolysates production.

Manothermosonication, high-pressure homogenization, and their combinations with pH-shifting improve the techno-functionality and digestibility of hemp protein

The growing interest in plant-based proteins, particularly hemp protein, has driven the exploration of new methods to address the limitations of its industrial utilization, such as its poor functional properties. This study examined how manothermosonication (MTS) and high-pressure homogenization (HPH) techniques, along with pH-shifting, could enhance the techno-functionality of hemp protein isolate (HPI) and produce functionalized hemp protein nanoaggregates. Optimal processing conditions of MTS were 50 °C, 200 kPa, and 90 s, as determined by response surface methodology. The pH-shifting + MTS and pH-shifting + HPH treatments reduced the size of hemp protein nanoaggregates by 3.2- and 1.9-fold, respectively, compared to the untreated HPI. The changes in secondary (FTIR, CD), tertiary (SH group, fluorescence intensity), and 3D structures (XRD) confirmed modifications of treated hemp proteins. The XRD patterns showed an increase in the %crystallinity values from 21% to 29% upon the pH-shifting + MTS treatment. The combination of pH-shifting + MTS resulted in the highest solubility and digestibility, 2.1- and 1.3-fold higher than the untreated HPI, respectively, followed by the MTS-alone and HPH treatments. In addition, the emulsion properties (emulsion activity, stability, creaming index, and droplet sizes) exhibited the highest values in HPI treated by pH-shifting + MTS and + HPH. The MTS and HPH technologies provide a promising viewpoint for producing protein nanoaggregates with enhanced functional properties for microencapsulation, edible film, and other applications.

Biotransformation approaches using solid-state fermentation and germination with high-intensity ultrasound to produce added-value hemp protein nanoaggregates

Hemp protein, with superior nutritional content and high digestibility, has attracted significant interest in recent years. However, like other plant protein sources, hemp protein has still suffered from underutilization in industrial applications due to its poor solubility attributes. To overcome this limitation, this study aimed to use biotechnological approaches such as germination (1, 3, 5 days) and solid-state fermentation using Lactobacillusplantarum and Bacillus subtilis to enhance the physicochemical, functional, and bioactive properties of hemp protein. The high-intensity ultrasound combined with these treatments for further modifications. The protein recovery in the defatted flour increased by 42% in germinated isolates compared to the untreated isolate. The primary structure changes were proven by degrading high molecular weight proteins to lower weights in SDS-PAGE. The secondary and tertiary structure changes of isolates were observed in FTIR, CD spectra, DSC, and free SH groups. While the particle sizes of isolates were reduced by 83%, solubility increased by 133% in fermented and ultrasonicated isolates. Water, oil absorption capacities and emulsion activities were increased by 103%, 194%, and 300%, respectively, in modified isolates compared to the untreated isolate. Total phenolic and antioxidant activities of isolates improved in germinated and fermented isolates. Fermentation, germination, and ultrasonication are promising, green, safe, alternative biomodification techniques that may effectively improve protein functionality. These clean-labeled proteins can also be used as bioactive agents in the food industry.

Sulfonamides stimulate ROS formation upon glycation of human carbonic anhydrase II

Advanced glycation end products are the most important species of glycation pathway, and cause disorders such as oxidative stress and diabetes. Sulfonamide compounds, which are generally known as widespread inhibitors, are potential agents used in different drug products, which can readily enter biological matrices. In the present work, the structure and activity of human carbonic anhydrase II studied in the presence of glucose as well as four sulfonamide agents from different views. These included enzyme kinetics, free lysine content, fluorescence spectroscopy, circular dichroism, and ROS measurement. Our results indicated that upon glycation, the structure of HCA II collapses and 8 to 13 lysine residues will be more available based on ligand incubation. Secondary and tertiary structural changes were also observed in the presence and absence of sulfonamide agents using fluorescence and circular dichroism methods, respectively. These spectroscopic data also showed a remarkable increase in hydrophobicity and decrease in α-helix contents during glycation, especially after 35 days of incubation. ROS assay was studied in the presence of glucose and sulfonamide compounds, that demonstrated the role of sulfonamide compounds in ROS formation in the presence of glucose in a synergistic manner. Overall, our data indicated that sulfonamides act as a stimulant factor upon prolonged interaction with HCA II and may intensify the complications of some disorders, such as diabetes and other conformational diseases.

Effect of non-covalently bound polyphenols on the structural and functional properties of wheat germ protein

This study investigated the effects of epigallocatechin-3-gallate (EGCG), chlorogenic acid (CA), piceatannol (PIC) and ferulic acid (FA) on the conformational and functional properties of wheat germ protein (WGP). The WGP-polyphenol interaction was confirmed by native polyacrylamide gel electrophoresis and detection of polyphenol content in the complex. Among the four polyphenols, EGCG showed the highest binding affinity to WGP. Moreover, FT-IR analysis indicated that the polyphenols changed the secondary structure of WGP, inducing an enhancement in alpha-helix content and a reduction in β-sheet structure. Fluorescence spectrum analysis demonstrated the presence of polyphenols induced tertiary structure change of WGP, leading to the deconstruction and unfolding of protein. In addition, the main interaction force for WGP-EGCG was hydrogen bonding and van der Waals forces, and for WGP-CA complexes was electrostatic interaction, while interaction force for WGP-PIC and WGP-FA complexes were hydrophobic interaction. Moreover, the addition of polyphenols significantly increased the absolute value of zeta potential while decreased surface hydrophobicity and free sulfhydryl content of WGP. The solubility and functionality of WGP were mainly improved by CA and EGCG. The findings in this study suggest that WGP-polyphenol complexes have the potential to be applied as novel functional food ingredients.

Hexametaphosphate, a Common Food Additive, Aggregated the Hen Egg White Lysozyme.

Polyphosphate polymers are chains of phosphate monomers chemically bonded together via phosphoanhydride bonds. They are found in all prokaryotic and eukaryotic organisms and are among the earliest, most anionic, and most mysterious molecules known. They are everywhere, from small cellular components to additives in our food. There is a strong association between hyperphosphatemia and mortality. That is why it is crucial to assess how polyphosphates, as food additives, affect the quality of edible proteins. This study investigated the effect of inexpensive and widely used food additives (hexametaphosphate labeled as E452) on bakery items, meat products, fish, and soft drinks. Using various spectroscopic and microscopic techniques, we examined how hexametaphosphate affected the aggregation propensity, structure, and stability of a commonly used food protein: hen egg white lysozyme (HEWL). The solubility of HEWL is affected in a bimodal fashion by the concentration of hexametaphosphate. The bimodal concentration-dependent effect was also observed in the tertiary and secondary structural changes. Hexametaphosphate-induced HEWL aggregates were amorphous, as evidenced by ThT fluorescence, far-UV CD, and TEM imaging. This study showed that the food additive (hexametaphosphate) may denature and aggregate proteins and may lead to undesirable health issues.

Effect of ultrasonic assisted grafting on the structural and functional properties of mung bean protein isolate conjugated with maltodextrin through maillard reaction

Four different methods of maillard reaction including ultrasound (150 W, 10 min) assisted, classical wet heating (80 °C, 60min), moderate water bath heating (60°C, 12 to 30 h) and dry state method (60 °C, 79 % relative humidity and 48 h) were used to Mung bean protein isolate – Maltodexrtin conjugates (MPI-MD) preparation. The samples prepared under ultrasound and wet heating were chosen for further analysis according to degree of graft and UV-absorbance at 420 nm. Higher glycosylation at short time and lower browning were obtained under ultrasound treatment. Covalent attachment in conjugates confirmed by SDS-polyacrylamide gel electrophoresis. The structural analysis revealed prominent unfolding effect of ultrasound waves on the protein's molecules. The decrease of α-helix content was related to the exposure of buried amino group residues during reaction. Glycation of MPI under ultrasound caused changes in tertiary structure of protein and leads to decrease in the fluorescence intensity compared with native and wet heating treatments. FTIR spectra confirmed the conjugation of the MPI and MD and suggested that protein structure was changed and ultrasound promoted the graft reaction more than wet heating treatment. Conjugated MPI showed higher emulsification and solubility index than MPI, moreover the effect of ultrasonic waves on ameliorated functional properties was impressive than those for wet heating treatment. Overall, this study showed use of ultrasonication in maillard reaction was a suitable method for producing MPI- MD conjugates and improved the efficiency of graft reaction and functional properties of grafts.

Mitigation of the Deleterious Effect of Heavy Metals on the Conformational Stability of Ubiquitin through Osmoprotectants.

The Ubiquitin-Proteasome System (UPS) is important in protein homeostasis and is involved in many cell processes. UPS's wide range of regulatory activities is based on the unique and diverse signals transmitted through all-encompassing processes. Cells need a fully functional UPP to cope with oxidative stress, so cellular redox status modulates ubiquitin activity. However, these protein quality control systems are compromised under adverse conditions such as heavy metal stress, resulting in pathological conditions. Heavy metals disrupt the physiological action of sensitive proteins by forming complexes with side-chain functional groups or by dislocating critical metal ions in metalloproteins. In addition, perturbation in the structure of Ubiquitin may affect the ubiquitin-proteasome pathway. In this study, it has been investigated the effects of heavy metals likewise chromium (Cr), cadmium (Cd), and mercury chloride (HgCl2) on the conformational stability of Ubiquitin as well as overcome their hazardous effect, the interaction of osmo-protectants such as Sesamol, gallic acid, Glycine, and ascorbic acid have also been explored in the study. The near and far UV-circular dichroism measurements deduced the secondary and tertiary structural changes. The size of the Ubiquitin before and after exposure to heavy metals was measured by DLS (dynamic light scattering). Docking research was also used to investigate the interaction of Ubiquitin with various heavy metals. Near and far UV-circular dichroism (CD) measurements revealed that mercury, chromium, and cadmium disrupt Ubiquitin's secondary and tertiary structure. The effect of chromium, even at low concentrations, was significantly deleterious compared to cadmium and mercury chloride. Ubiquitin's far-UV circular dichroism spectra subjected to heavy metals were recorded in several osmo-protectants, such as ascorbic acid, Glycine, gallic acid, and Sesamol, which offset the adverse effects of heavy metals. DLS studies revealed a noteworthy change in the hydrodynamic radius of Ubiquitin in the presence of heavy metals. Docking analysis revealed a significant binding affinity of mercury and cadmium ions with Ubiquitin. This study can infer the heavy metals' disruption of Ubiquitin's secondary and tertiary structure. Osmo-protectants produced by animal cells are more effective against heavy metals than plant antioxidants.

Effect of Palmitic Acid on Tertiary Structure of Glycated Human Serum Albumin

Non-enzymatic glycation is a process, which can be best described as a significant posttranslational modification of various proteins. It emerges in hyperglycemic conditions and may have an impact on albumin stability as well as its activity and physical and chemical properties, essentially affecting all its physiological functions. The goal of this research was to answer the following questions: (i) how does the glycation of defatted human serum albumin by glucose–fructose syrup (GFS) alter its tertiary structure; (ii) does palmitic acid (PA), a component of palm oil, affect the in vitro glycation process and cause conformational changes of glycated albumin; and (iii) does PA inhibit the formation of Advanced Glycation End-Products (AGEs)? Therefore, in order to point out differences in the tertiary structure of macromolecules, the absorption and emission of fluorescence spectra and their second derivatives, excitation fluorescence and synchronous spectra, Red-Edge Excitation Shift (REES effect), and the degree of modification of sulfhydryl groups of defatted, non-glycated (HSA) and glycated human serum albumin (gHSA) with GFS and glycated with GFS and PA were investigated. In the present study, it has been confirmed that the glycation of albumin in the presence of GFS and PA causes changes in both HSA and gHSA tertiary structures, respectively. Moreover, palmitic acid, at ratios of 1.5:1 and 3:1 with glycated albumin, does not exhibit inhibition of AGEs formation. This study indicates the fact that the structural changes, especially those of glycated albumin, are important for treatment planning because the type of the interaction between the components and their primary transporter may be altered as the disease progresses or in the elderly.

Improving AlphaFold2-based protein tertiary structure prediction with MULTICOM in CASP15

Since the 14th Critical Assessment of Techniques for Protein Structure Prediction (CASP14), AlphaFold2 has become the standard method for protein tertiary structure prediction. One remaining challenge is to further improve its prediction. We developed a new version of the MULTICOM system to sample diverse multiple sequence alignments (MSAs) and structural templates to improve the input for AlphaFold2 to generate structural models. The models are then ranked by both the pairwise model similarity and AlphaFold2 self-reported model quality score. The top ranked models are refined by a novel structure alignment-based refinement method powered by Foldseek. Moreover, for a monomer target that is a subunit of a protein assembly (complex), MULTICOM integrates tertiary and quaternary structure predictions to account for tertiary structural changes induced by protein-protein interaction. The system participated in the tertiary structure prediction in 2022 CASP15 experiment. Our server predictor MULTICOM_refine ranked 3rd among 47 CASP15 server predictors and our human predictor MULTICOM ranked 7th among all 132 human and server predictors. The average GDT-TS score and TM-score of the first structural models that MULTICOM_refine predicted for 94 CASP15 domains are ~0.80 and ~0.92, 9.6% and 8.2% higher than ~0.73 and 0.85 of the standard AlphaFold2 predictor respectively.

Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit

Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/β complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impβ as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T= 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impβ-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.