A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine.

In the present work, biophysical insight into the binding interactions of the protein, hen egg white (HEW) lysozyme (Lyz) with an anticancer drug, 6-mercaptopurine (6-MP)' was investigated by using a combination of spectroscopic and computational tools. 6-MP, a synthetic analog of natural purines, is a well-known anticancer drug and antiviral agent that inhibits the synthesis of RNA, DNA, and proteins. Lysozyme is a single-chain protein that can combine with endogenous and exogenous substances to exert its antiviral, antibacterial, and antitumor effects. The intrinsic fluorescence of lysozyme was quenched with the increased addition of 6-MP. The quenching mechanism was found to be static in nature as shown by the fluorescence lifetime and excitation spectrum measurements. The conformational changes of Lyz in the presence of 6-MP were monitored both at the ensemble and single-molecule level by using synchronous fluorescence spectroscopy, circular dichroism (CD), and fluorescence correlation spectroscopy (FCS). Molecular docking results predicted the probable binding sites for 6-MP on Lyz. The experimental findings are in good agreement with the results obtained by the molecular dynamics (MD) simulation study. Graphical abstract.

Phytoconstituents of Ashwagandha as potential inhibitors of human islet amyloid polypeptide (hIAPP): an in silico investigation.

Amylin or human islet amyloid polypeptide (hIAPP) is a small peptide co-secreted with insulin. Its peripheral aggregation on the lipid bilayer leads to fibril formation. The formation of hIAPP fibrils is hypothesized to rupture the membrane of ß -cells, which culminates in ß-cell death. Following additional studies, amylin fibril formation is a hallmark of T2DM and is also implicitly responsible for Alzheimer's disease. This study reports the virtual screening of 1000 phytoconstituents of traditional Indian medicinal plants to get potential inhibitors of amylin, which will likely restrict and block amyloid aggregation, preventing the progression of T2DM and Alzheimer's illness. The compounds having drug-likeness properties (acquired from ADMET calculations) and highest binding affinities (from molecular docking) are subjected to molecular dynamics (MD) simulation to investigate the temporal stability of the conformations of the complexes. This study discovers that Withaferin A and Withacoagulin have the highest binding affinity for amylin, and their stability with amylin was verified further by parameters such as RMSD, RMSF, number of H-bonds and MMPBSA. Individual principle component analysis (PCA) confirms the stable complex formation of amylin with Withaferin A and Withacoagulin. We strongly believe that wet-lab experiments and clinical trials will help to validate our computational findings.Communicated by Ramaswamy H. Sarma.

Interaction of vitamin B12 with ß-lactoglobulin: a computational study.

The ß-Lactoglobulin (ßLG) is a major whey protein that has the potential to bind various ligands; hence it is used as a model protein in protein-ligand interaction studies. Vitamin B12 is an essential nutrient for the human body, which helps in the synthesis of DNA, proteins, and the production of red blood cells. Binding interaction of vitamin B12 with ßLG will help to understand the potency of ßLG as a transporter for vitamin B12. Our experimental findings already showed that ßLG binds with vitamin B12 successfully (Swain et al., 2020). Nevertheless, to further support our experimental results firmly, here, we have employed computational tools such as molecular docking and molecular dynamics (MD) simulation. The molecular docking technique was used to elucidate the probable binding sites and binding affinity of vitamin B12 on ßLG. The docked complex of vitamin B12 with ßLG was subjected to MD simulation to investigate its stability and other interaction properties over a time frame. The study revealed that the compound is stable, and vitamin B12 imposes no change to the secondary structure of the ßLG. The computational results agree reasonably well with our experimental study.

In silico investigation of spice molecules as potent inhibitor of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel infectious disease that is in rapid growth. Several trials are going on worldwide to find a solution for this pandemic. The viral replication can be blocked by inhibiting the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (SARS-CoV-2 RBD Spro) and the SARS-CoV-2 main protease (SARS-CoV-2 Mpro). The binding of potential small molecules to these proteins can inhibit the replication and transcription of the virus. The spice molecules that are used in our food have antiviral, antifungal and antimicrobial properties. As spice molecules are consumed in the diet, hence its antiviral properties against SARS-CoV-2 will benefit in a significant manner. Therefore, in this work, the molecular docking of 30 selected spice molecules (screened through ADME property) was performed to identify the potential inhibitors for the RBD Spro and Mpro of SARS-CoV-2. We have found that though all the molecules bind actively with the SARS-CoV-2 RBD Spro and Mpro, but Piperine has the highest binding affinity among the 30 screened molecules. Besides, the comparative study between Piperine and currently used drugs show that Piperine is more effective. The interaction of Piperine with RBD Spro and Mpro is further validated by the molecular dynamics (MD) simulation studies. The free energy landscape and binding free energy results also, support for the stable complex formation of Piperine with RBD Spro and Mpro. We anticipate immediate wet-lab experiments and clinical trials in support of this computational study that might help to inhibit the SARS-CoV-2 virus. Communicated by Ramaswamy H. Sarma.

Conformational dynamics of myoglobin in the presence of vitamin B12: A spectroscopic and in silico investigation.

Myoglobin is an essential transport protein of heart and muscle tissues that acts as a local oxygen reservoir and a marker in different diseased conditions. On the other hand, Vitamin B12 is a vital nutrient that helps synthesize red blood cells, DNA, and proteins. To understand the ability of vitamin B12 to bind to the excess of myoglobin produced in the body under certain conditions (muscle injuries, severe trauma, etc.), it is essential to dig into the interaction between them. Therefore, the present study reports the binding interaction of vitamin B12 and myoglobin employing different spectroscopic and computational methods. The myoglobin's intrinsic fluorescence is quenched by vitamin B12 via static nature as observed from steady-state as well as time-resolved fluorescence measurements. The microenvironment of myoglobin's tryptophan residue gets affected, but there is no change observed in its α-helical content by vitamin B12 as seen from synchronous fluorescence and circular dichroism measurements. The probable binding of vitamin B12 on myoglobin was elucidated through molecular docking, and the interaction stability was studied by molecular dynamics simulation. The determination of vitamin B12's affinity to myoglobin and its effect on the conformational transitions of myoglobin might afford valuable insight for clinical pharmacology.

Spectroscopic and computational insight into the conformational dynamics of hemoglobin in the presence of vitamin B12.

Protein-ligand interactions play a significant role in all living organisms, thereby affecting the design and application of drugs and other biomaterials. The current study reports the binding of vitamin B12 to hemoglobin, employing optical spectroscopy and computational methods. It is observed that vitamin B12 quenched the intrinsic fluorescence of hemoglobin. The nature of quenching appears to be static according to the steady-state and time-resolved fluorescence measurements. The conformational changes of hemoglobin caused by vitamin B12 interactions were studied by synchronous fluorescence spectroscopy and protein secondary structure analyses. The synchronous fluorescence spectra indicate the tryptophan residue microenvironment change while no secondary structural change is observed from circular dichroism spectra and molecular dynamics (MD) simulation study. The computational molecular docking elucidated the probable binding of vitamin B12 at the active site of hemoglobin, whereas the stability of the hemoglobin-vitamin B12 complex was studied by MD simulation. The study might be helpful for the treatment of pernicious anemia, hereditary transcobalamin deficiency, and performance enhancement of elite athletes.

Spectroscopic insight into the interaction of dopamine with spherical gold nanoparticles.

Dopamine (DA) is a monoamine neurotransmitter of phenethylamine and catecholamine families, which is present in the central nervous system (CNS) and its periphery. Since DA is associated with several functions in the brain and body (motivational salience, reward, motor control, paracrine messenger, etc.), any imbalance in the DA level can trigger several neurodegenerative and other diseases. On the other hand, the spherical gold nanoparticles (AuNPs) can be used for drug delivery in several parts of the body. In addition, AuNPs also have the potentiality to penetrate through the blood-brain barrier and interact with the central nervous system without causing any toxicity. In view of many applications, it is important to look into the interaction between DA and AuNPs for a potential drug delivery model in DA related diseases. Here, we have used the steady-state and time-resolved fluorescence spectroscopic tools to investigate the binding interaction of DA with AuNPs. The nature of the quenching mechanism was confirmed through both steady-state and time-resolved fluorescence measurements. The binding constants along with the number of binding sites were estimated from the steady-state fluorescence measurements. The distance between DA and AuNPs was calculated using Förster's theory to verify the possibility of fluorescence resonance energy transfer (FRET) from DA to AuNPs.

Biophysical study on complex formation between ß-Lactoglobulin and vitamin B12.

Biophysical insight into the binding interaction between the major whey protein, ß-Lactoglobulin (ßLG) and vitamin B12, was studied using different spectroscopic tools such as steady-state & time-resolved fluorescence spectroscopy, Circular Dichroism (CD) and Fluorescence Correlation Spectroscopy (FCS). The intrinsic fluorescence of ßLG was quenched by vitamin B12. From the time-resolved fluorescence experiment, the nature of quenching was found to be static suggesting ground-state complex formation between ßLG and vitamin B12, which was also supported by the excitation spectra. Synchronous fluorescence spectra revealed that the tryptophan residue microenvironment of ßLG was affected by the vitamin B12. The CD spectra suggested that the secondary structure of the ßLG remains unaffected by vitamin B12. From the FCS experiment, the tertiary structure of ßLG was observed to be stable in the presence of vitamin B12 at the single-molecule level. The outcome of this study might have potential applications in the food and pharmaceutical industry.

A simulation study on the influence of energy migration and relative interaction strengths of homo- and hetero-FRET on the net FRET efficiency.

Förster resonance energy transfer (FRET) is a powerful method for probing biomolecular conformations and dynamics in bulk as well as at a single-molecule level. FRET utilizes non-radiative mechanisms to transfer energy between fluorophores, donor and acceptor when placed in close proximity. The FRET efficiency has a strong distance dependence and serves as a direct read-out for molecular interaction. In case of a significant overlap of donor emission and absorption spectra, the excited state energy can be exchanged between the identical donors in close proximity, which eventually migrates back and forth until it gets dissipated. This form of energy transfer is called energy migration or homo-FRET. Here, we have simulated FRET efficiency by considering the donor-donor interaction strength (ξDD) and donor-acceptor interaction strength (ξDA) under conditions of non-uniform distribution of molecules. Our earlier studies indicate that energy migration modulate the FRET efficiency for various values of ξDD and ξDA. We, therefore, determined the limiting values of acceptor concentration (CLA) that will allow the determination of FRET efficiency in the absence and presence of energy migration. Taken together, our study optimizes the conditions for meaningful FRET efficiency for a given FRET pair for better reporting of molecular interactions.

Stability of ß-turn in LaR2C-N7 peptide for its translation-inhibitory activity against hepatitis C viral infection: A molecular dynamics study.

Hepatitis C virus (HCV) requires an essential host factor, human La protein, for its translation and replication activity. Earlier, it was demonstrated that a 24-mer synthetic peptide (LaR2C) encompassing residues 112 to 184 of the natural human La protein interacts with the HCV internal ribosome entry site (IRES) and inhibits translation. Interestingly, a shorter version of the same LaR2C peptide, LaR2C-N7, containing residues 174 to 180 (KYKETDL), with a unique ß-turn secondary structure, is sufficient to inhibit IRES mediated translation of HCV. Hence, it is imperative to understand the role of each amino acid of this heptapeptide towards ß-turn formation which will then help in designing potential drugs against HCV infection. Here, we use Nanoscale Molecular Dynamics (NAMD) simulation to investigate the factors modulating its ß-turn formation and stability. Using 100 ns simulation paradigms, we find that the peptide populated the type 1 ß-turn conformation in its free form in solution. However, simulation of the single-site mutants of the heptapeptide revealed that none of the 7 mutants retained the ß-turn conformation with sufficient stability. We observed that the ß-turn was stabilized mainly by the side chain interaction, salt-bridge and weak hydrogen bonds between K3 and D6 residues. Y2, K1 and K3 sites upon mutation heavily destabilized the ß-turn when compared to alteration at the E4 and T5 sites which would then drastically reduce its HCV RNA IRES binding capabilities. Taken together, our results provide a basis for designing peptidomimetics as potential anti-HCV drug candidates.