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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.

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.

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 β-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 β-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 β-turn formation and stability. Using 100 ns simulation paradigms, we find that the peptide populated the type 1 β-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 β-turn conformation with sufficient stability. We observed that the β-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 β-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.

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.

Forster 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.