Isothermal Titration Calorimetric Results Research Articles

Surfactin-like lipopeptides from Bacillus clausii efficiently bind to spike glycoprotein of SARS-CoV-2

The coronavirus disease 2019 (COVID-19) rapidly spread across the globe, infecting millions and causing hundreds of deaths. It has been now around three years but still, it remained a serious threat worldwide, even after the availability of some vaccines. Bio-surfactants are known to have antiviral activities and might be a potential alternative for the treatment of SARS-CoV-2 infection. In the present study, we have isolated and purified, a surfactin-like lipopeptide produced by a probiotic bacterial strain Bacillus clausii TS. Upon purification and characterization with MALDI analysis, the molecular weight of the lipopeptide is confirmed as 1037 Da (similar to surfactin C) which is known to have antiviral activities against various enveloped viruses. Purified surfactin-like lipopeptide showed efficient binding and inhibition of SARS-CoV-2 spike (S1) protein, revealed by competitive ELISA assay. Further, we have explored the complete thermodynamics of the inhibitory binding of surfactin-like lipopeptide with S1 protein using isothermal titration calorimetric (ITC) assay. ITC results are in agreement with ELISA with a binding constant of 1.78 × 10−4 M−1. For further validation of the inhibitory binding of surfactin-like lipopeptide with S1 protein and its receptor binding domain (RBD), we performed molecular docking, dynamics, and simulation experiments. Our results suggested that surfactin could be a promising drug agent for the spike protein targeting drug development strategy against SARS-CoV-2 and other emerging variants.Communicated by Ramaswamy H. Sarma

How black tea pigment theaflavin dyes chicken eggs: Binding affinity study of theaflavin with ovalbumin

Theaflavin (TF), which is the key pigment in black tea, is a health-promoting food component with beneficial effects on humans. However, the interactions by which these effects are transferred and exerted into protein-rich foods are unclear. Here, egg ovalbumin (OVA) was selected as a representative dietary protein to ascertain their binding mechanism. Steady-state, time-resolved fluorescence and isothermal titration calorimetric results showed that TF can interact well with OVA with an affinity magnitude of 104. The noncovalent binding was mainly driven by hydrophobic interaction and hydrogen bonds. Structural analysis displayed that the TF binding pocket significantly overlapped with one of the surrounding specific IgE-binding epitopes, thereby causing a subtle structural adjustment on the secondary conformation of OVA. The biological complexation model that was delineated here will help understand how black tea dyes egg white in tea egg products and for the development of protein-rich carriers in functional foods.

Identification of the binding between three fluoronucleoside analogues and fat mass and obesity-associated protein by isothermal titration calorimetry and spectroscopic techniques

In this work, the interactions between fat mass and obesity-associated (FTO) protein and three fluoronucleoside analogues (three-member-ring compound (1a), five-member-ring compound (1b) and six-member-ring compound (1c)) have been investigated by fluorescence, ultraviolet-visible absorption spectroscopy, isothermal titration calorimetric (ITC) and molecular modeling. Analysis of fluorescence data showed that the binding between three analogues and FTO occurred via a static quenching mechanism. Both ITC and fluorescence results indicated that 1b is the strongest quencher. In contrast to spectroscopy techniques, ITC results suggested that there is no binding for 1c. ITC results showed that the binding between FTO and 1a (or 1b) were exothermic. Fluorescence results showed that the binding between three analogues and FTO were endothermic. Results of thermodynamic analysis and molecular modeling suggested that it was entropy driven event between FTO and 1a (or 1b).

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.

Cysteine as a potential anti-amyloidogenic agent with protective ability against amyloid induced cytotoxicity

Protein aggregation and misfolding have been allied with numerous human disorders and thus inhibition of such occurrence has been center for intense research efforts against these diseases. Here, we investigated anti-fibrillation activity of cysteine and its effect on kinetics of stem bromelain amyloid fibril formation. We established the anti-fibrillation and anti aggregation activities of cysteine by using multiple approaches like turbidity measurements, dye binding assays (ThT and ANS) and structural changes were monitored by circular dichroism (CD) followed by electron microscopy. Our experimental study inferred that cysteine inhibits temperature induced fibrillation of protein in a concentration dependent way. In addition, MDA-MB-231 cell viability of pre-formed amyloid was increased in presence of cysteine as compared to the fibrils alone. Furthermore, dynamic light scattering studies of native, aggregated as well as incubated (amyloids in presence of cysteine) samples indicates that cysteine restores native like structures of stem bromelain. Isothermal titration calorimetric results revealed that hydrogen bonding between cysteine and stem bromelain plays a significant role during inhibition of stem bromelain aggregation. However, thiophilic interaction between thiol group of cysteine and aromatic amino acid residue of stem bromelain may also have noteworthy role in inhibition of amyloid formation.

Anesthetic 2,2,2-trifluoroethanol induces amyloidogenesis and cytotoxicity in human serum albumin

Trifluoroethanol (TFE) mimics the membrane environments as it simulates the hydrophobic environment and better stabilizes the secondary structures in peptides owing to its hydrophobicity and hydrogen bond-forming properties. Its dielectric constant approximates that of the interior of proteins and is one-third of that of water. Human serum albumin (HSA) is a biological transporter. The effect of TFE on HSA gives the clue about the conformational changes taking place in HSA on transport of ligands across the biological membranes. At 25% (v/v) and 60% (v/v) TFE, HSA exhibits non-native β-sheet, altered tryptophan fluorescence, exposed hydrophobic clusters, increased thioflavin T fluorescence and prominent red shifted Congo red absorbance, and large hydrodynamic radii suggesting the aggregate formation. Isothermal titration calorimetric results indicate weak binding of TFE and HSA. This suggests that solvent-mediated effects dominate the interaction of TFE and HSA. TEM confirmed prefibrillar at 25% (v/v) and fibrillar aggregates at 60% (v/v) TFE. Comet assay of prefibrillar aggregates showed DNA damage causing cell necrosis hence confirming cytotoxic nature. On increasing concentration of TFE to 80% (v/v), HSA showed retention of native-like secondary structure, increased Trp and ANS fluorescence, a transition from β-sheet to α-helix. Thus, TFE at high concentration possess anti- aggregating potency.

Drug–protein interactions in micellar media: Thermodynamic aspects

Devising directions for surfactant assisted effective controlled release of drugs requires a quantitative and qualitative understanding of the drug–protein, drug–surfactant, and surfactant–protein interactions. In this work, the effect of micellar environment on the binding of naproxen and diclofenac sodium with bovine serum albumin has been studied. The isothermal titration calorimetric (ITC) results suggest that the binding of naproxen is reduced with the protein when it is delivered from micellar media. However, the binding is observed to be strengthened for diclofenac sodium. The differential scanning calorimetric results suggest that the integrity of the binding sites is not altered under the employed micellar conditions. The ITC results further suggest that the numbers of naproxen and diclofenac sodium molecules partitioning/binding per micelle of HTAB are 15 and 38, respectively. In the micelles, naproxen is restricted to the surface of the micelles whereas diclofenac sodium is able to partition in the palisade layers. A detailed understanding of the energetics of the drug–protein interactions under different conditions helps in devising directions for effective drug delivery. The ITC and DSC results have shown that the micelles assisted drug–protein interactions are modified depending on the hydrophobic content of the drug.

The C-terminal variable domain of LigB fromLeptospiramediates binding to fibronectin

Adhesion through microbial surface components that recognize adhesive matrix molecules is an essential step in infection for most pathogenic bacteria. In this study, we report that LigB interacts with fibronectin (Fn) through its variable region. A possible role for LigB in bacterial attachment to host cells during the course of infection is supported by the following observations: (i) binding of the variable region of LigB to Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner reduces the adhesion of Leptospira, (ii) inhibition of leptospiral attachment to Fn by the variable region of LigB, and (iii) decrease in binding of the variable region of LigB to the MDCK cells in the presence of Fn. Furthermore, we found a significant reduction in binding of the variable region of LigB to Fn using small interfering RNA (siRNA). Finally, the isothermal titration calorimetric results confirmed the interaction between the variable region of LigB and Fn. This is the first report to demonstrate that LigB binds to MDCK cells. In addition, the reduction of Fn expression in the MDCK cells, by siRNA, reduced the binding of LigB. Taken together, the data from the present study showed that LigB is a Fn-binding protein of pathogenic Leptospira spp. and may play a pivotal role in Leptospira-host interaction during the initial stage of infection.

Unusually Strong Binding to the DNA Minor Groove by a Highly Twisted Benzimidazole Diphenylether: Induced Fit and Bound Water

RT29 is a dicationic diamidine derivative that does not obey the classical "rules" for shape and functional group placement that are expected to result in strong binding and specific recognition of the DNA minor groove. The compound contains a benzimidazole diphenyl ether core that is flanked by the amidine cations. The diphenyl ether is highly twisted and gives the entire compound too much curvature to fit well to the shape of the minor groove. DNase I footprinting, fluorescence intercalator displacement studies, and circular dichroism spectra, however, indicate that the compound is an AT specific minor groove binding agent. Even more surprisingly, quantitative biosensor-surface plasmon resonance and isothermal titration calorimetric results indicate that the compound binds with exceptional strength to certain AT sequences in DNA with a large negative enthalpy of binding. Crystallographic results for the DNA complex of RT29 compared to calculated results for the free compound show that the compound undergoes significant conformational changes to enhance its minor groove interactions. In addition, a water molecule is incorporated directly into the complex to complete the compound-DNA interface, and it forms an essential link between the compound and base pair edges at the floor of the minor groove. The calculated DeltaCp value for complex formation is substantially less than the experimentally observed value, which supports the idea of water being an intrinsic part of the complex with a major contribution to the DeltaCp value. Both the induced fit conformational changes of the compound and the bound water are essential for strong binding to DNA by RT29.

Aggregation Behavior and Thermodynamics of Binding between Poly(ethylene oxide)-block-Poly(2-(diethylamino)ethyl methacrylate) and Plasmid DNA

The aggregation behavior and the thermodynamics of binding between poly(ethylene oxide)-block-poly(2-(diethylamino)ethyl methacrylate) (PEO-b-PDEAEMA) block copolymers and plasmid DNA were examined. Binding between the polymer and DNA were confirmed by gel electrophoresis. The high affinity between the polymer and DNA was demonstrated through the ethidium bromide (EtBr) displacement assay, and the binding was found to be related to the stoichiometric balance between the amine group of the polymer and the DNA nucleotide molar ratio (N/P molar ratio). The light scattering and TEM results showed that, at low polymer concentration, the hydrodynamic radii (R(h)) of the polymer/DNA complexes was around 90 nm; however, at sufficiently high polymer concentration, the complexes condensed to around 35 nm induced by a structural rearrangement of the amphiphilic nature of the block copolymer. The isothermal titration calorimetric results showed that the binding between the polymer and DNA is driven by a large favorable enthalpy.

Binding of 8-Anilinonaphthalene Sulfonate to Dimeric and Tetrameric Concanavalin A: Energetics and Its Implications on Saccharide Binding Studied by Isothermal Titration Calorimetry and Spectroscopy

The binding of 8-anilinonaphthalene sulfonate to concanavalin A has been investigated. Isothermal titration calorimetry (ITC) and circular dichroism studies have been performed under different experimental conditions to understand the binding quantitatively and evaluate contributions of different forces responsible for it. Isothermal titration calorimetric results of concanavalin A with ANS at pH 5.2 and 2.5, where it exists as a dimer, indicated binding heterogeneity and two classes of noninteracting sites. Enhancement of the binding constants from native to pH 2.5 suggests that the ANS binding is strongly influenced by the protein charge and the favorable alteration in the structure of concanavalin A as suggested by near-UV CD results. No binding was observed with the tetrameric form of concanavalin A, indicating shielding of sites due to dimerization of canonical dimers. The results have also demonstrated existence of a hydrophobic binding site that is distinct from the saccharide binding site.

Does the anesthetic 2,2,2‐trifluoroethanol interact with bovine serum albumin by direct binding or by solvent‐mediated effects? A calorimetric and spectroscopic investigation

Thermal unfolding of bovine serum albumin (BSA) has been studied in the presence of 2,2,2-trifluoroethanol (TFE) using high-sensitivity microdifferential scanning calorimetry. Quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. TFE is observed to be a stabilizer or a destabilizer of the folded state of BSA depending on the pH. CD spectroscopy revealed an increase in the alpha-helical content of BSA and a decrease in the tertiary structure in the presence of increasing molalities of TFE. Isothermal titration calorimetric results do not indicate appreciable binding of the TFE molecules to BSA. TFE quenches the steady-state tryptophan fluorescence of BSA only at higher molalities and there is no effect on the tryptophan fluorescence at lower molalities. The calorimetric and spectroscopic results obtained in this work suggest that solvent-mediated effects dominate the interaction of TFE with BSA and the binding component may be very weak. Since the binding component is very weak, one of the possibilities of anesthetic action of TFE molecules on the actual targets may be through perturbation of the structural and dynamic properties of the lipid bilayer so that the function of crucial but unspecified membrane proteins is affected.