Insight into the binding of PEG-400 with eye protein alpha-crystallin: Multi spectroscopic and computational approach: possible therapeutics targeting eye diseases.

Alpha-crystallin (α-crystallin) is an important eye protein having chaperone activity; its aggregation and precipitation are vital in cataract development. Polyethylene glycol-400 (PEG-400) is an important constituent of eye drops and artificial tears. The present study was targeted to study the binding of α-crystallin and PEG-400 employing multi spectroscopy, isothermal titration calorimetry (ITC) along with molecular modeling and docking approach. There was an apparent hypochromism in α-crystallin in the presence of varying PEG-400 concentrations; the binding constant obtained was 0.9 X 105 M-1 implying that strong binding is taking place between α-crystallin and PEG-400. Fluorescence spectroscopy suggested good binding of PEG-400 to α-crystallin with a binding constant (K) of 106 M-1. Moreover, fluorescence quenching studies carried out at three different temperatures suggested α-crystallin-PEG-400 complex formation to be guided by combination of static and dynamic modes. Thermodynamic parameters suggested α-crystallin-PEG-400 complex formation is driven by van der Waals forces and hydrogen bonding, making it seemingly specific. Far UV-CD spectra revealed no shift in the peak implying no alterations in the secondary structure of α-crystallin upon PEG-400 binding further validating complex formation. In vitro assays were further entrenched by in silico assays. Molecular modeling was used to make the functionally active form of α-crystallin. A binding pocket located in the ß chain was delineated by Prank Web; molecular docking showed binding of PEG-400 in this pocket. This study will give an insight into the binding of PEG-400 with α-crystallin and can serve as a rationale for the discovery of therapeutic molecules that can be used for the treatment of eye-related crystallin-directed diseases. HighlightsPEG-400 is a constituent of lubricant eye drops.Molecular modelling gave functionally active crystallin.Molecular docking showed PEG-400 in the binding pocket.Fluorescence binding revealed good bindingα-crystallin and PEG400.The α-crystallin-PEG-400 complex was guided by static and dynamic quenching.Communicated by Ramaswamy H. Sarma.

Crowding Milleu stabilizes apo-myoglobin against chemical-induced denaturation: Dominance of hardcore repulsions in the heme devoid protein.

Macromolecular crowding can have significant consequences on the structure and dynamics of a protein. The size and shape of a co-solute molecule and the nature of protein contribute significantly in macromolecular crowding, which results in different outcomes in similar conditions. The structure of apo-myoglobin (apo-Mb) both in the absence and presence of denaturants (GdmCl and urea) was investigated in crowded conditions at pH 7.0, with a comparable size of crowders (~70 kDa) but of different shapes (ficoll and dextran) at various concentrations using spectroscopic techniques like absorption and circular dichroism to monitor changes in secondary and tertiary structure, respectively. The crowders in the absence of denaturants showed structural stabilization of the tertiary structure while no significant change in the secondary structure was observed. The effect of crowders on the stability of the protein was also investigated using probes such as Δε291 and θ222 using chemical denaturants. The analysis of chemical-induced denaturation curves showed that both the crowders stabilize apo-Mb by increasing the values of the midpoint of transition (Cm) and change in free energy in the absence of denaturant (∆GD°), and it was observed that dextran 70 shows more stabilization than ficoll 70 under similar conditions. In this study apo-Mb showed stabilization under crowded conditions, which is a deviation from earlier work from our group where holo form of the same protein was destabilized. This study emphasizes that volume exclusion is a dominant force in a simple protein while soft interactions may play important role in the proteins that are possessing prosthetic group. Hence, the effect of crowders is protein-dependent, and excluded volume plays a great role in the stabilization of apo-Mb, which does not interact with the crowders.

Heparin Accelerates the Protein Aggregation via the Downhill Polymerization Mechanism: Multi-Spectroscopic Studies to Delineate the Implications on Proteinopathies.

Heparin is one of the members of the glycosaminoglycan (GAG) family, which has been associated with protein aggregation diseases including Alzheimer's disease, Parkinson's disease, and prion diseases. Here, we investigate heparin-induced aggregation of bovine serum albumin (BSA) using different spectroscopic techniques [absorption, 8-anilino-1-naphthalene sulfonic acid (ANS) and thioflavin T (ThT) fluorescence binding, and far- and near-UV circular dichroism]. Kinetic measurements revealed that heparin is involved in the significant enhancement of aggregation of BSA. The outcomes showed dearth of the lag phase and a considerable change in rate constant, which provides conclusive evidence, that is, heparin-induced BSA aggregation involves the pathway of the downhill polymerization mechanism. Heparin also causes enhancement of fluorescence intensity of BSA significantly. Moreover, heparin was observed to form amyloids and amorphous aggregates of BSA which were confirmed by ThT and ANS fluorescence, respectively. Circular dichroism measurements exhibit a considerable change in the secondary and tertiary structure of the protein due to heparin. In addition, binding studies of heparin with BSA to know the cause of aggregation, isothermal titration calorimetry measurements were exploited, from which heparin was observed to promote the aggregation of BSA by virtue of electrostatic interactions between positively charged amino acid residues of protein and negatively charged groups of GAG. The nature of binding of heparin with BSA is very much apparent with an appreciable heat of interaction and is largely exothermic in nature. Moreover, the Gibbs free energy change (ΔG) is negative, which indicates spontaneous nature of binding, and the enthalpy change (ΔH) and entropy change (ΔS) are also largely negative, which suggest that the interaction is driven by hydrogen bonding.

Guidelines and Safety Considerations in the Laboratory Diagnosis of SARS-CoV-2 Infection: A Prerequisite Study for Health Professionals.

Coronavirus disease 2019 (COVID-19) is an emerging challenging area for the researchers to buckle up against the spread and control of the virus. Since earlier times, the diagnosis has been an important procedure in estimating the fate of epidemics by indicating the extent to which disease has been spread and to the extent, further disease prognosis would occur. The absence of anti-viral therapies and vaccines for COVID-19 at present suggests early diagnosis and isolation of the patients as the only smart approach available as of now. Presently, the increasing death rates, faster rates of transmission, non-availability of vaccines, and treatment have over-pressurized the researchers, health professionals, and government officials to develop effective clinical strategies in diagnosis and to come up with guidelines to be followed during conduction of each diagnostic procedure for maintaining healthcare systems. Since the incubation period of this virus is 2-14 days, a patient can transmit the infection without showing symptoms. Therefore, early diagnosis and isolation of susceptible individuals are the only way to limit the spread of the virus. Significance of diagnosis and triaging, information on specimen collection, safety considerations while handling, transport, and storage of samples have been highlighted in this paper to make people more aware and develop better clinical strategies in the future.

Identification of natural compounds as potent inhibitors of SARS-CoV-2 main protease using combined docking and molecular dynamics simulations.

Coronavirus disease 2019 (COVID-19) has emerged from China and globally affected the entire population through the human-to-human transmission of a newly emerged virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genome of SARS-CoV-2 encodes several proteins that are essential for multiplication and pathogenesis. The main protease (Mpro or 3CLpro) of SARS-CoV-2 plays a central role in its pathogenesis and thus is considered as an attractive drug target for the drug design and development of small-molecule inhibitors. We have employed an extensive structure-based high-throughput virtual screening to discover potential natural compounds from the ZINC database which could inhibit the Mpro of SARS-CoV-2. Initially, the hits were selected on the basis of their physicochemical and drug-like properties. Subsequently, the PAINS filter, estimation of binding affinities using molecular docking, and interaction analyses were performed to find safe and potential inhibitors of SARS-CoV-2 Mpro. We have identified ZINC02123811 (1-(3-(2,5,9-trimethyl-7-oxo-3-phenyl-7H-furo[3,2-g]chromen-6-yl)propanoyl)piperidine-4-carboxamide), a natural compound bearing appreciable affinity, efficiency, and specificity towards the binding pocket of SARS-CoV-2 Mpro. The identified compound showed a set of drug-like properties and preferentially binds to the active site of SARS-CoV-2 Mpro. All-atom molecular dynamics (MD) simulations were performed to evaluate the conformational dynamics, stability and interaction mechanism of Mpro with ZINC02123811. MD simulation results indicated that Mpro with ZINC02123811 forms a stable complex throughout the trajectory of 100 ns. These findings suggest that ZINC02123811 may be further exploited as a promising scaffold for the development of potential inhibitors of SARS-CoV-2 Mpro to address COVID-19.

Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders.

Generally, in vivo function and structural changes are studied by probing proteins in a dilute solution under in vitro conditions, which is believed to be mimicking proteins in intracellular milieu. Earlier, thermal-induced denaturation of myoglobin, in the milieu of crowder molecule showed destabilization of the metal protein. Destabilization of protein by thermal-induced denaturation involves a large extrapolation, so, the reliability is questionable. This led us to measure the effects of macromolecular crowding on its stability by chemical-induced denaturation of the protein using probes like circular dichroism and absorption spectroscopy in the presence of dextran 70 and ficoll 70 at various pHs (acidic: 6.0, almost neutral:7.0 and basic: 8.0). Observations showed that the degree of destabilization of myoglobin was greater due to ficoll 70 as compared to that of dextran 70 so it can be understood that the nature of the crowder or the shape of the crowder has an important role towards the stability of proteins. Additionally, the degree of destabilization was observed as pH dependent, however the pH dependence is different for different crowders. Furthermore, isothermal titration calorimetry and molecular docking studies confirmed that both the crowders (ficoll and dextran) bind to heme moiety of myoglobin and a single binding site was observed for each.

Macromolecular crowding induces molten globule state in the native myoglobin at physiological pH.

Here, we report the formation of molten globule state of the native myoglobin in crowded environment. We have used Soret absorption spectroscopy and far-UV circular dichroism to monitor changes in tertiary and secondary structures of myoglobin, respectively. Our results reveal that in the presence of ficoll 70, the secondary structure of myoglobin remains unchanged while tertiary structure is lost significantly. 1-anilinonaphthalene-8-sulfonate binding experiments showed that myoglobin in the presence of various concentrations of ficoll 70, has newly exposed hydrophobic surfaces. Dynamic light scattering measurements show that there is almost 1.5 times increase in the hydrodynamic volume of myoglobin in the crowded environment. These structural characteristics of myoglobin in the presence of 300mg/ml ficoll 70 resemble those of molten globule state. Isothermal titration calorimetric (ITC) measurements show that ficoll 70 binds to myoglobin, whereas it shows no interaction with apo form of the protein. ITC results indicate that the reason behind this unique behavior of ficoll 70 towards myoglobin may be interaction of ficoll 70 with the heme group of myoglobin, which was further confirmed by the docking studies. We hypothesize that the soft interactions between heme and ficoll 70 leads to the formation of molten globule in myoglobin.