Integrated spectroscopic and computational analyses unravel the molecular interaction of pesticide azinphos-methyl with bovine beta-lactoglobulin.

Organophosphorus are typically hazardous chemicals used in the pharmaceutical, agricultural, and other industries. They pose a serious risk to human life and can be fatal upon direct exposure. Hence, studying the interaction between such compounds with proteins is crucial for environmental, health, and food safety. In this study, we investigated the interaction mechanism between azinphos-methyl (AZM) and ß-lactoglobulin (BLG) at pH 7.4 using a combination of biophysical techniques. Intrinsic fluorescence investigations revealed that BLG fluorescence was quenched in the presence of increasing AZM concentrations. The quenching mechanism was identified as static, as evidenced by a decrease in the fluorescence quenching constant (1.25 × 104, 1.18 × 104, and 0.86 × 104 M-1) with an increase in temperatures. Thermodynamic calculations (ΔH > 0; ΔS > 0) affirmed the formation of a complex between AZM and BLG through hydrophobic interactions. The BLG's secondary structure was found to be increased due to AZM interaction. Ultraviolet -visible spectroscopy data showed alterations in BLG conformation in the presence of AZM. Molecular docking highlighted the significant role of hydrophobic interactions involving residues such as Val43, Ile56, Ile71, Val92, Phe105, and Met107 in the binding between BLG and AZM. A docking energy of -6.9 kcal mol-1, and binding affinity of 1.15 × 105 M-1 suggest spontaneous interaction between AZM and BLG with moderate to high affinity. These findings underscore the potential health risks associated with the entry of AZM into the food chain, emphasizing the need for further consideration of its impact on human health.

Probing the interaction mechanisms between sunset yellow dye and trypsin protein leading to amorphous aggregation under low pH conditions.

Protein aggregation poses a significant concern in the field of food sciences, and various factors, such as synthetic food dyes, can contribute to protein aggregation. One such dye, Sunset Yellow (SY), is commonly employed in the food industry. Trypsin was used as a model protein to assess the impact of SY. We employed several biophysical techniques to examine the binding and aggregation mechanisms between SY and trypsin at different pHs. Results from intrinsic fluorescence measurements indicate a stronger interaction between SY and trypsin at pH 2.0 compared to pH 6.0. Turbidity data reveal trypsin aggregation in the presence of 0.05-3.0 mM SY at pH 2.0, while no aggregation was observed at pH 6.0. Kinetic data demonstrate a rapid, lag-phase-free SY-induced aggregation of trypsin. Circular dichroism analysis reveals that trypsin adopts a secondary structure in the presence of SY at pH 6.0, whereas at pH 2.0, the secondary structure was nearly lost with increasing SY concentrations. Furthermore, turbidity and kinetics data suggest that trypsin aggregation depends on trypsin concentrations and pH. Our study highlights potential health risks associated with the consumption of SY, providing insights into its impact on human health and emphasizing the necessity for further research in this field.

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.

SDBS induces multiple catalase conformations in a dose-dependent manner.

Amyloid fibrils have been linked to several incurable diseases. They are long and thin fibrous proteins that self-assemble into fibrils. Small molecules can stimulate amyloid fibrillation, but the mechanism by which this happens is not well understood. This study examined how a negatively charged benzene ring containing surfactant, sodium dodecylbenzene sulphonate (SDBS), affects the fibrillation of bovine liver catalase (BLC). After SDBS treatment, BLC conformational changes were examined in vitro using turbidity, RLS kinetics, intrinsic fluorescence, ThT fluorescence, far-UV CD, and TEM. BLC in the native state was alpha-helical at pH 7.4, while it was converted to a random coil structure at pH 2.0. Far-UV CD and intrinsic fluorescence data showed that at concentrations <0.1 mM of SDBS, randomly coiled BLC assumed a native-like alpha-helical structure. However, between 0.1 and 1.0 mM SDBS, BLC was aggregated. ThT fluorescence and far-UV CD measurements showed the amyloid-like structures in the aggregated BLC. At higher SDBS concentrations (>1.0 mM) at pH 2.0, BLC again attains a native-like alpha-helical structure. It is essential for therapeutic purposes to clearly understand the process underlying surfactant- or lipid-induced fibrillation.

Characterization of pH-induced conformational changes in recombinant DENV NS2B-NS3pro.

The increasing frequency of Dengue is a cause of severe epidemics and therefore demands strategies for effective prevention, diagnosis, and treatment. DENV-protease is being investigated as a potential therapeutic target. However, due to the flat and highly charged active site of the DENV-protease, designing orthosteric medicines is very difficult. In this study, we have done a thorough analysis of pH-dependent conformational changes in recombinantly expressed DENV protease using various spectroscopic techniques. Our spectroscopic study of DENV protease (NS2B-NS3pro) at different pH conditions gives important insights into the dynamicity of structural conformation. At physiological pH, the DENV-protease exists in a random-coiled state. Lowering the pH promotes the formation of alpha-helical and beta-sheet structures i.e. gain of secondary structure as shown by Far-UV CD. The light scattering and Thioflavin T (ThT)-binding assay proved the aggregation-prone tendency of DENV-protease at pH 4.0. Further, the confocal microscopy image intensity showed the amorphous aggregate formation of DENV protease at pH 4.0. Thus, the DENV protease acquires different conformations with changes in pH conditions. Together, these results have the potential to facilitate the design of a conformation destabilizer-based therapeutic strategy for dengue fever.

Bovine liver catalase turns into three conformational states after exposure to an anionic surfactant.

The mechanism by which anionic surfactants promote amyloid fibril is not well understood. Here, we investigated how sodium dodecyl sulfate (SDS), a negatively charged surfactant, affects the fibrillation of the partially unfolded random-coiled bovine liver catalase (BLC) at a pH of 2.0. We used several methods, including turbidity, RLS kinetics, intrinsic fluorescence, ThT fluorescence, far-UV CD, and TEM imaging, to evaluate the conformational changes of BLC in vitro in response to SDS treatment. BLC is a multimeric protein and well folded at physiological pH but forms a random coil structure at pH 2.0. Intrinsic fluorescence and far-UV CD data showed that below 0.1 mM SDS, random coiled BLC turned into a native-like structure. BLC incubated with an SDS concentration ranging from 0.1 to 2.0 mM led to the formation of aggregates. The ThT fluorescence intensity was enhanced in the aggregated BLC samples (0.1-2.0 mM SDS), and cross beta-sheeted structure was detected by the far UV CD measurements. BLC adopts a complete alpha-helical structure upon interacting with SDS at a more than 2.0 mM concentration at pH 2.0. Understanding the mechanism of surfactant- or lipid-induced fibrillation is important for therapeutic purposes.

Anionic surfactant causes dual conformational changes in insulin.

Amyloid fibrillation is a process by which proteins aggregate and form insoluble fibrils that are implicated in several neurodegenerative diseases. In n this study, we aimed to investigate the impact of the negatively charged detergent sodium dodecyl sulfate (SDS) on insulin amyloid fibrillation at pH 7.4 and 2.0, as SDS has been linked to the acceleration of amyloid fibrillation in vitro, but the underlying molecular mechanism is not fully understood. Our findings show that insulin forms amyloid-like aggregates in the presence of SDS at concentrations ranging from 0.05 to 1.8 mM at pH 2.0, while no aggregates were observed at SDS concentrations greater than 1.8 mM, and insulin remained soluble. However, at pH 7.4, insulin remained soluble regardless of the concentration of SDS. Interestingly, the aggregated insulin had a cross-ß sheet secondary structure, and when incubated with higher SDS concentrations, it gained more alpha-helix. The electrostatics and hydrophobic interaction of SDS and insulin may contribute to amyloid induction. Moreover, the SDS-induced aggregation was not affected by the presence of salts. Furthermore, as the concentration of SDS increased, the preformed insulin amyloid induced by SDS began to disintegrate. Overall, our study sheds light on the mechanism of surfactant-induced amyloid fibrillation in insulin protein.

Interaction Mechanism between α-Lactalbumin and Caffeic Acid: A Multispectroscopic and Molecular Docking Study.

Caffeic acid (CA) is a phenolic acid found in a variety of foods. In this study, the interaction mechanism between α-lactalbumin (ALA) and CA was explored with the use of spectroscopic and computational techniques. The Stern-Volmer quenching constant data suggest a static mode of quenching between CA and ALA, depicting a gradual decrease in quenching constants with temperature rise. The binding constant, Gibbs free energy, enthalpy, and entropy values at 288, 298, and 310 K were calculated, and the obtained values suggest that the reaction is spontaneous and exothermic. Both in vitro and in silico studies show that hydrogen bonding is the dominant force in the CA-ALA interaction. Ser112 and Lys108 of ALA are predicted to form three hydrogen bonds with CA. The UV-visible spectroscopy measurements demonstrated that the absorbance peak A280nm increased after addition of CA due to conformational change. The secondary structure of ALA was also slightly modified due to CA interaction. The circular dichroism (CD) studies showed that ALA gains more α-helical structure in response to increasing concentration of CA. The surface hydrophobicity of ALA is not changed in the presence of ethanol and CA. The present findings shown herein are helpful in understanding the binding mechanism of CA with whey proteins for the dairy processing industry and food nutrition security.

Iron response elements (IREs)-mRNA of Alzheimer's amyloid precursor protein binding to iron regulatory protein (IRP1): a combined molecular docking and spectroscopic approach.

The interaction between the stem-loop structure of the Alzheimer's amyloid precursor protein IRE mRNA and iron regulatory protein was examined by employing molecular docking and multi-spectroscopic techniques. A detailed molecular docking analysis of APP IRE mRNA∙IRP1 reveals that 11 residues are involved in hydrogen bonding as the main driving force for the interaction. Fluorescence binding results revealed a strong interaction between APP IRE mRNA and IRP1 with a binding affinity and an average binding sites of 31.3 × 106 M-1 and 1.0, respectively. Addition of Fe2+(anaerobic) showed a decreased (3.3-fold) binding affinity of APP mRNA∙IRP1. Further, thermodynamic parameters of APP mRNA∙IRP1 interactions were an enthalpy-driven and entropy-favored event, with a large negative ΔH (-25.7 ± 2.5 kJ/mol) and a positive ΔS (65.0 ± 3.7 J/mol·K). A negative ΔH value for the complex formation suggested the contribution of hydrogen bonds and van der Waals forces. The addition of iron increased the enthalpic contribution by 38% and decreased the entropic influence by 97%. Furthermore, the stopped-flow kinetics of APP IRE mRNA∙IRP1 also confirmed the complex formation, having the rate of association (kon) and the rate of dissociation (koff) as 341 µM-1 s-1, and 11 s-1, respectively. The addition of Fe2+ has decreased the rate of association (kon) by ~ three-fold, whereas the rate of dissociation (koff) has increased by ~ two-fold. The activation energy for APP mRNA∙IRP1 complex was 52.5 ± 2.1 kJ/mol. The addition of Fe2+ changed appreciably the activation energy for the binding of APP mRNA with IRP1. Moreover, circular dichroism spectroscopy has confirmed further the APP mRNA∙IRP1 complex formation and IRP1 secondary structure change with the addition of APP mRNA. In the interaction between APP mRNA and IRP1, iron promotes structural changes in the APP IRE mRNA∙IRP1 complexes by changing the number of hydrogen bonds and promoting a conformational change in the IRP1 structure when it is bound to the APP IRE mRNA. It further illustrates how IRE stem-loop structure influences selectively the thermodynamics and kinetics of these protein-RNA interactions.

Agitation does not induce fibrillation in reduced hen egg-white lysozyme at physiological temperature and pH.

Several proteins and peptides tend to form an amyloid fibril, causing a range of unrelated diseases, from neurodegenerative to certain types of cancer. In the native state, these proteins are folded and soluble. However, these proteins acquired ß-sheet amyloid fibril due to unfolding and aggregation. The conversion mechanism from well-folded soluble into amorphous or amyloid fibril is not well understood yet. Here, we induced unfolding and aggregation of hen egg-white lysozyme (HEWL) by reducing agent dithiothreitol and applied mechanical sheering force by constant shaking (1000 rpm) on the thermostat for 7 days. Our turbidity results showed that reduced HEWL rapidly formed aggregates, and a plateau was attained in nearly 5 h of incubation in both shaking and non-shaking conditions. The turbidity was lower in the shaking condition than in the non-shaking condition. The thioflavin T binding and transmission electron micrographs showed that reduced HEWL formed amorphous aggregates in both conditions. Far-UV circular dichroism results showed that reduced HEWL lost nearly all alpha-helical structure, and ß-sheet secondary structure was not formed in both conditions. All the spectroscopic and microscopic results showed that reduced HEWL formed amorphous aggregates under both conditions.

SDS induces amorphous, amyloid-fibril, and alpha-helical structures in the myoglobin in a concentration-dependent manner.

Amyloid fibrils have been linked to a number of diseases. Surfactants imitate plasma membrane lipids and induce amyloid fibrils. This study examined the effects of the anionic surfactant sodium dodecyl sulfate (SDS) at pH 4.5 on equine skeletal muscle myoglobin (E-Mb). To analyze the effect of SDS on aggregation and amyloid-fibril formation to E-Mb, we used various spectroscopic techniques (turbidity, light scattering, intrinsic fluorescence, ThT fluorescence, and circular dichroism (CD)), electrophoretic, and microscopic techniques. Turbidity, SDS-PAGE, and light scattering all indicated the formation of E-Mb aggregates at SDS concentrations ranging from 0.2 mM to 1.0 mM. In the presence of 0.4 mM SDS, far-UV CD and TEM data indicate that E-MB forms amorphous aggregates. ThT binding, Far-UV CD, and TEM findings indicate that E-Mb forms amyloid-like structures in the presence of 0.6-1.0 mM SDS. However, no aggregation was seen at SDS concentrations above 1 mM. In the presence of high SDS concentrations (> 1 mM), the E-Mb exhibited native-like α-helical structure. As a result, SDS exhibited three distinct behaviors: amorphous aggregates, amyloid-fibrils, and helix-inducer. These findings also shed light on how amyloid fibrils are formed when anionic surfactants are introduced, which is a significant takeaway.

Molecular mechanism of insulin aggregation in the presence of a cationic surfactant.

Protein aggregation and amyloid fibrillation are connected with neurodegenerative disorders. Insulin, a small molecular weight protein related to type II diabetes, has been shown to self-assemble to form protein aggregates. In this work, we investigated the effects of cetyltrimethylammonium bromide (CTAB) of insulin on the in vitro aggregation process at pH 7.4 and 2.0. The aggregation tendency of insulin was measured using a variety of biophysical approaches, including turbidity measurements, light scattering, far UV-CD, ThT dye binding, and transmission electron microscopy. The turbidity results demonstrated that at pH 7.4, a low concentration of CTAB (30-180 µM) causes insulin aggregation but at higer concentration (>180 µM) aggregation was not seen. However, at pH 2.0, both low as well as high concentrations of CTAB were unable to promote insulin aggregation. The ThT dye binding and far-UV CD data suggest that aggregation induced by CTAB is not having an ordered structure. Insulin treated with higher concentrations (>180 µM) of CTAB, the insulin gained a secondary structure. The possible cause of inducing aggregation in insulin is electrostatic and hydrophobic interaction because insulin contains a net negative charge at pH 7.4 and no aggregation at pH 2.0 due to electrostatic repulsion.

Molecular insight into the modulation of ovalbumin fibrillation by allura red dye at acidic pH.

The synthetic food additive dye induces amyloid fibrillation has many implications in the laboratory and industries. The effect of Allura red (AR), on the fibrillation of ovalbumin (Ova) at pH 2.0 was investigated. The influence of salt and pH was also seen on AR-induced Ova aggregation. We have used several spectroscopic and microscopy techniques to characterize the changes. The turbidity data suggest that concentrations above 0.05 mM of AR induce aggregation, and the size of aggregates increased in response to AR concentration. The kinetics data showed that the AR induces Ova aggregation quickly without lag time. The aggregates induced by AR have amyloid-like aggregates confirmed by far-UV CD and TEM. NaCl has very marginal effects in AR-induced aggregation. The turbidity results clearly state that Ova is not forming aggregates with pH above 4.0 due to electrostatic repulsion. However, Ova forms bigger aggregates in the presence of 0.5 mM AR at a pH below 4.0. These spectroscopic data suggest that the amyloid fibrillation that occurs in Ova is due to electrostatic and hydrophobic interaction. The amyloid fibrillation induced by AR dye in protein should be taken seriously for food safety purposes.

Screening and identification of potential MERS-CoV papain-like protease (PLpro) inhibitors; Steady-state kinetic and Molecular dynamic studies.

MERS-CoV belongs to the coronavirus group. Recent years have seen a rash of coronavirus epidemics. In June 2012, MERS-CoV was discovered in the Kingdom of Saudi Arabia, with 2,591 MERSA cases confirmed by lab tests by the end of August 2022 and 894 deaths at a case-fatality ratio (CFR) of 34.5% documented worldwide. Saudi Arabia reported the majority of these cases, with 2,184 cases and 813 deaths (CFR: 37.2%), necessitating a thorough understanding of the molecular machinery of MERS-CoV. To develop antiviral medicines, illustrative investigation of the protein in coronavirus subunits are required to increase our understanding of the subject. In this study, recombinant expression and purification of MERS-CoV (PLpro), a primary goal for the development of 22 new inhibitors, were completed using a high throughput screening methodology that employed fragment-based libraries in conjunction with structure-based virtual screening. Compounds 2, 7, and 20, showed significant biological activity. Moreover, a docking analysis revealed that the three compounds had favorable binding mood and binding free energy. Molecular dynamic simulation demonstrated the stability of compound 2 (2-((Benzimidazol-2-yl) thio)-1-arylethan-1-ones) the strongest inhibitory activity against the PLpro enzyme. In addition, disubstitutions at the meta and para locations are the only substitutions that may boost the inhibitory action against PLpro. Compound 2 was chosen as a MERS-CoV PLpro inhibitor after passing absorption, distribution, metabolism, and excretion studies; however, further investigations are required.

An insight into the binding and inhibition of eye ζ-crystallin by the environmental toxin arsenic: implications in eye diseases.

Arsenic contamination is highly prevalent in food chain, soil and groundwater. Continuous exposure to elevated levels of this environmental toxin is a global concern. Studies have reported enriched accumulation of arsenic in the eyes compared to other body organs leading to various eye diseases. Here, the impact of arsenic exposure on the enzymatic eye ζ-crystallin has been investigated. Arsenic inhibited the activity of the enzyme with an IC50 value of 35 µM. It decreased the free thiol group content of ζ-crystallin due to protein oxidation. The binding of arsenic with ζ-crystallin was explored using biophysical and computational tools. The enzyme undergoes some conformational changes upon arsenic binding. The binding constant (Kb) was determined to be 1.2 × 102 M-1. Thermodynamic parameters were determined by isothermal titration calorimetry (ITC) and the binding energy (ΔG) was calculated to be -3.52 kcal/mol. Molecular docking studies helped in visualizing the amino acid residues (especially Cys165) of the enzyme involved in binding with arsenic. Continuous arsenic exposure is expected to increase the eye crystallin-related abnormalities, elevating the risk of cataractogenesis. Therefore, proper measures need to be taken by authorities to control the contamination of arsenic in the environment and groundwater.Communicated by Ramaswamy H. Sarma.

Elucidation of kinetic and structural properties of eye lens ζ-crystallin: an in vitro and in silico approach.

The Arabian Camelus dromedarius contains significant concentration of eye lens ζ-crystallin. This enzyme is also present in other life forms including humans, however in lower catalytic amounts. The recombinant camel ζ-crystallin was expressed in the E. coli BL21 (DE3) pLysS strain and purified using HisTrap column. The Km of the enzyme for 9,10-phenanthrenequinone (9,10-PQ) substrate and NADPH cofactor was determined to be 11.66 and 50.93 µM, respectively. The Vmax for 9,10-PQ and NADPH was obtained as 23.19 and 19.98 µM min-1, respectively. The optimum activity of the purified enzyme was found to be at pH 6.0 and at 55 °C. Different physico-chemical parameters were analysed including instability index (II), aliphatic index (AI) and the GRAVY index to establish proper characterization. The sequence of the recombinant ζ-crystallin was subjected to homology modelling using SWISS-MODEL webserver followed by validation of the modelled target structure. The evaluation of the modelled ζ-crystallin was performed by several parameters including Ramachandran plot, Z-score values followed by molecular dynamics (MD) simulation. The cumulative analysis of the physico-chemical, quantitative, qualitative and the essential dynamics of simulation of ζ-crystallin and its complexes with 9,10-PQ and NADPH helped in verifying the acceptable quality and stability of the ζ-crystallin structure.Communicated by Ramaswamy H. Sarma.

Role of salts and solvents on the defibrillation of food dye "sunset yellow" induced hen egg white lysozyme amyloid fibrils.

Several food dyes are known to induce amyloid fibrillation when interacting with proteins. Here, we studied the role of sunset yellow (SY) in the amyloid fibrillation of hen egg white lysozyme (HEWL) and characterized the changes using spectroscopy techniques. Turbidity results showed that SY dye induces aggregation in HEWL in concentrations dependent manner. The aggregation induced by SY dye is kinetically very fast, no lag phase was detected, and the kinetics process follows an isodesmic kinetics pathway. The SY-dye induce aggregates have cross-ß secondary structure confirmed by far-UV CD measurements. The effect of salts and solvents was also seen on SY-induced aggregates. Turbidity, far-UV CD, and kinetics results suggest that certain concentrations of NaCl and (NH4)2SO4 solubilize the SY-induce amyloid fibrils, but (NH4)2SO4 is more effective. Similarly, solvents are also solubilized the SY-induces HEWL amyloid fibrillation but the order of defibrillation is as follows: Isopropanol> ethanol > methanol which signified that isopropanol is more effective than other solvents. The salts and solvents data suggest that the electrostatic, as well as hydrophobic interaction, is responsible for SY-induced amyloid fibrillation. These conformational changes should be examined, more seriously for the purpose of food safety.

Implications of Diabetes-Induced Altered Metabolites on Retinal Neurodegeneration.

Diabetic retinopathy (DR) is one of the major complications of diabetic eye diseases, causing vision loss and blindness worldwide. The concept of diabetic retinopathy has evolved from microvascular disease into more complex neurovascular disorders. Early in the disease progression of diabetes, the neuronal and glial cells are compromised before any microvascular abnormalities clinically detected by the ophthalmoscopic examination. This implies understanding the pathophysiological mechanisms at the early stage of disease progression especially due to diabetes-induced metabolic alterations to damage the neural retina so that early intervention and treatments options can be identified to prevent and inhibit the progression of DR. Hyperglycemia has been widely considered the major contributor to the progression of the retinal damage, even though tight control of glucose does not seem to have a bigger effect on the incidence or progression of retinal damage that leads to DR. Emerging evidence suggests that besides diabetes-induced hyperglycemia, dyslipidemia and amino acid defects might be a major contributor to the progression of early neurovascular retinal damage. In this review, we have discussed recent advances in the alterations of key metabolites of carbohydrate, lipid, and amino acids and their implications for neurovascular damage in DR.

Encapsulation of MERS antigen into α-GalCer-bearing-liposomes elicits stronger effector and memory immune responses in immunocompetent and leukopenic mice.

Objectives: Here, we prepared a liposome-based vaccine formulation containing Middle East Respiratory Syndrome Coronavirus papain-like protease (MERS-CoV-PLpro). Methods: A persistent leukopenic condition was induced in mice by injecting cyclophosphamide (CYP) three days before each dose of immunization. Mice were immunized on days 0, 14 and 21 with α-GalCer-bearing MERS-CoV PLpro-encapsulated DPPC-liposomes (α-GalCer-MERS-PLpro-liposomes or MERS-CoV PLpo-encapsulated DPPC-liposomes (MERS-PLpro-liposomes), whereas the antigen emulsified in Alum (MERS-PLpro-Alum) was taken as a control. On day 26, the blood was taken from the immunized mice to analyze IgG titer, whereas the splenocytes were used to analyze the lymphocyte proliferation and the level of cytokines. In order to assess the memory immune response, mice were given a booster dose after 150 days of the last immunization. Results: The higher levels of MERS-CoV-PLpro-specific antibody titer, IgG2a and lymphocyte proliferation were noticed in mice immunized with α-GalCer-MERS-PLpro-liposomes. Besides, the splenocytes from mice immunized with α-GalCer-MERS-PLpro-liposomes produced larger amounts of IFN-γ as compared to the splenocytes from MERS-PLpro-liposomes or MERS- PLpro-Alum immunized mice. Importantly, an efficient antigen-specific memory immune response was observed in α-GalCer-MERS-PLpro-liposomes immunized mice. Conclusions: These findings suggest that α-GalCer-MERS-PLpro-liposomes may substantiate to be a successful vaccine formulation against MERS-CoV infection, particularly in immunocompromised individuals.

Modulation of the Structure and Stability of Novel Camel Lens Alpha-Crystallin by pH and Thermal Stress.

Alpha-crystallin protein performs structural and chaperone functions in the lens and comprises alphaA and alphaB subunits at a molar ratio of 3:1. The highly complex alpha-crystallin structure challenges structural biologists because of its large dynamic quaternary structure (300−1000 kDa). Camel lens alpha-crystallin is a poorly characterized molecular chaperone, and the alphaB subunit possesses a novel extension at the N-terminal domain. We purified camel lens alpha-crystallin using size exclusion chromatography, and the purity was analyzed by gradient (4−12%) sodium dodecyl sulfate−polyacrylamide gel electrophoresis. Alpha-crystallin was equilibrated in the pH range of 1.0 to 7.5. Subsequently, thermal stress (20−94 °C) was applied to the alpha-crystallin samples, and changes in the conformation and stability were recorded by dynamic multimode spectroscopy and intrinsic and extrinsic fluorescence spectroscopic methods. Camel lens alpha-crystallin formed a random coil-like structure without losing its native-like beta-sheeted structure under two conditions: >50 °C at pH 7.5 and all temperatures at pH 2.0. The calculated enthalpy of denaturation, as determined by dynamic multimode spectroscopy at pH 7.5, 4.0, 2.0, and 1.0 revealed that alpha-crystallin never completely denatures under acidic conditions or thermal denaturation. Alpha-crystallin undergoes a single, reversible thermal transition at pH 7.5. The thermodynamic data (unfolding enthalpy and heat capacity change) and chaperone activities indicated that alpha-crystallin does not completely unfold above the thermal transition. Camels adapted to live in hot desert climates naturally exhibit the abovementioned unique features.