Solvent-Induced Lag Phase during the Formation of Lysozyme Amyloid Fibrils Triggered by Sodium Dodecyl Sulfate: Biophysical Experimental and In Silico Study of Solvent Effects.

Amyloid aggregates arise from either the partial or complete loss of the native protein structure or the inability of proteins to attain their native conformation. These aggregates have been linked to several diseases, including Alzheimer's, Parkinson's, and lysozyme amyloidosis. A comprehensive dataset was recently reported, demonstrating the critical role of the protein's surrounding environment in amyloid formation. In this study, we investigated the formation of lysozyme amyloid fibrils induced by sodium dodecyl sulfate (SDS) and the effect of solvents in the medium. Experimental data obtained through fluorescence spectroscopy revealed a notable lag phase in amyloid formation when acetone solution was present. This finding suggested that the presence of acetone in the reaction medium created an unfavorable microenvironment for amyloid fibril formation and impeded the organization of the denatured protein into the fibril form. The in silico data provided insights into the molecular mechanism of the interaction between acetone molecules and the lysozyme protofibril, once acetone presented the best experimental results. It was observed that the lysozyme protofibril became highly unstable in the presence of acetone, leading to the complete loss of its ß-sheet conformation and resulting in an open structure. Furthermore, the solvation layer of the protofibril in acetone solution was significantly reduced compared to that in other solvents, resulting in fewer hydrogen bonds. Consequently, the presence of acetone facilitated the exposure of the hydrophobic portion of the protofibril, precluding the amyloid fibril formation. In summary, our study underscores the pivotal role the surrounding environment plays in influencing amyloid formation.

A Computational-Experimental Investigation of the Molecular Mechanism of Interleukin-6-Piperine Interaction.

Herein, we elucidate the biophysical aspects of the interaction of an important protein, Interleukin-6 (IL6), which is involved in cytokine storm syndrome, with a natural product with anti-inflammatory activity, piperine. Despite the role of piperine in the inhibition of the transcriptional protein NF-κB pathway responsible for activation of IL6 gene expression, there are no studies to the best of our knowledge regarding the characterisation of the molecular interaction of the IL6-piperine complex. In this context, the characterisation was performed with spectroscopic experiments aided by molecular modelling. Fluorescence spectroscopy alongside van't Hoff analyses showed that the complexation event is a spontaneous process driven by non-specific interactions. Circular dichroism aided by molecular dynamics revealed that piperine caused local α-helix reduction. Molecular docking and molecular dynamics disclosed the microenvironment of interaction as non-polar amino acid residues. Although piperine has three available hydrogen bond acceptors, only one hydrogen-bond was formed during our simulation experiments, reinforcing the major role of non-specific interactions that we observed experimentally. Root mean square deviation (RMSD) and hydrodynamic radii revealed that the IL6-piperine complex was stable during 800 ns of simulation. Taken together, these results can support ongoing IL6 drug discovery efforts.

Experimental Approaches and Computational Modeling of Rat Serum Albumin and Its Interaction with Piperine.

The bioactive piperine (1-piperoyl piperidine) compound found in some pepper species (Piper nigrum linn and Piper sarmentosum Roxb) has been shown to have therapeutic properties and to be useful for well-being. The tests used to validate these properties were performed in vitro or with small rats. However, in all these assays, the molecular approach was absent. Although the first therapeutic trials relied on the use of rats, no proposal was mentioned either experimentally or computationally at the molecular level regarding the interaction between piperine and rat serum albumin (RSA). In the present study, several spectroscopic techniques were employed to characterize rat serum albumin and, aided by computational techniques, the protein modeling was proposed. From the spectroscopic results, it was possible to estimate the binding constant (3.9 × 104 M-1 at 288 K) using the Stern-Volmer model and the number of ligands (three) associated with the protein applying interaction density function model. The Gibbs free energy, an important thermodynamic parameter, was determined (-25 kJ/mol), indicating that the interaction was spontaneous. This important set of experimental results served to parameterize the computational simulations. The results of molecular docking and molecular dynamics matched appropriately made it possible to have detailed microenvironments of RSA accessed by piperine.

A flaw in applying the FRET technique to evaluate the distance between ligands and tryptophan residues in human serum albumin: Proposal of correction.

Due to its primordial function as a drug carrier, human serum albumin (HSA) is extensively studied regarding its binding affinity with developing drugs. Förster resonance energy transfer (FRET) is frequently applied as a spectroscopic molecular ruler to measure the distance between the binding site and the ligand. In this work, we have shown that most of the published results that use the FRET technique to estimate the distance from ligands to the binding sites do not corroborate the crystallography data. By comparing the binding affinity of dansyl-proline with HSA and ovotransferrin, we demonstrated that FRET explains the quenching provoked by the interaction of ligands in albumin. So, why does the distance calculation via FRET not corroborate the crystallography data? We have shown that this inconsistency is related to the fact that a one-to-one relationship between donor and acceptor is not present in most experiments. Hence, the quenching efficiency used for calculating energy transfer depends on distance and binding constant, which is inconsistent with the correct application of FRET as a molecular ruler. We have also shown that the indiscriminate attribution of 2/3 to the relative orientation of transition dipoles of the acceptor and donor (κ2) generates inconsistencies. We proposed corrections based on the experimental equilibrium constant and theoretical orientation of transition dipoles to correct the FRET results.

Unravelling the Interaction of Piperlongumine with the Nucleotide-Binding Domain of HSP70: A Spectroscopic and In Silico Study.

Piperlongumine (PPL) is an alkaloid extracted from several pepper species that exhibits anti-inflammatory and anti-carcinogenic properties. Nevertheless, the molecular mode of action of PPL that confers such powerful pharmacological properties remains unknown. From this perspective, spectroscopic methods aided by computational modeling were employed to characterize the interaction between PPL and nucleotide-binding domain of heat shock protein 70 (NBD/HSP70), which is involved in the pathogenesis of several diseases. Steady-state fluorescence spectroscopy along with time-resolved fluorescence revealed the complex formation based on a static quenching mechanism. Van't Hoff analyses showed that the binding of PPL toward NBD is driven by equivalent contributions of entropic and enthalpic factors. Furthermore, IDF and Scatchard methods applied to fluorescence intensities determined two cooperative binding sites with Kb of (6.3 ± 0.2) × 104 M-1. Circular dichroism determined the thermal stability of the NBD domain and showed that PPL caused minor changes in the protein secondary structure. Computational simulations elucidated the microenvironment of these interactions, showing that the binding sites are composed mainly of polar amino acids and the predominant interaction of PPL with NBD is Van der Waals in nature.

Detailed Characterization of the Cooperative Binding of Piperine with Heat Shock Protein 70 by Molecular Biophysical Approaches.

In this work, for the first time, details of the complex formed by heat shock protein 70 (HSP70) independent nucleotide binding domain (NBD) and piperine were characterized through experimental and computational molecular biophysical methods. Fluorescence spectroscopy results revealed positive cooperativity between the two binding sites. Circular dichroism identified secondary conformational changes. Molecular dynamics along with molecular mechanics Poisson Boltzmann surface area (MM/PBSA) reinforced the positive cooperativity, showing that the affinity of piperine for NBD increased when piperine occupied both binding sites instead of one. The spontaneity of the complexation was demonstrated through the Gibbs free energy (∆G < 0 kJ/mol) for different temperatures obtained experimentally by van't Hoff analysis and computationally by umbrella sampling with the potential of mean force profile. Furthermore, the mean forces which drove the complexation were disclosed by van't Hoff and MM/PBSA as being the non-specific interactions. In conclusion, the work revealed characteristics of NBD and piperine interaction, which may support further drug discover studies.

The Cytokine IL-1ß and Piperine Complex Surveyed by Experimental and Computational Molecular Biophysics.

The bioactive piperine, a compound found in some pepper species, has been widely studied because of its therapeutic properties that include the inhibition of an important inflammation pathway triggered by interleukin-1 beta (IL-1ß). However, investigation into the molecular interactions between IL-1ß and piperine is not reported in the literature. Here, we present for the first time the characterisation of the complex formed by IL-1ß and piperine through experimental and computational molecular biophysical analyses. Fluorescence spectroscopy unveiled the presence of one binding site for piperine with an affinity constant of 14.3 × 104 M-1 at 298 K. The thermodynamic analysis indicated that the interaction with IL-1ß was spontaneous (∆G = -25 kJ/mol) and, when split into enthalpic and entropic contributions, the latter was more significant. Circular dichroism spectroscopy showed that piperine did not affect IL-1ß secondary structure (~2%) and therefore its stability. The set of experimental data parameterized the computational biophysical approach. Through molecular docking, the binding site micro-environment was revealed to be composed mostly by non-polar amino acids. Furthermore, molecular dynamics, along with umbrella sampling, are in agreement with the thermodynamic parameters obtained by fluorescence assays and showed that large protein movements are not present in IL-1ß, corroborating the circular dichroism data.

Details of the cooperative binding of piperlongumine with rat serum albumin obtained by spectroscopic and computational analyses.

Piperlongumine (PPL) has presented a variety of important pharmacological activities. In recent pharmacokinetics studies in rats, this molecule reached 76.39% of bioavailability. Although PPL is present in the bloodstream, no information is found on the interaction between PPL and rat serum albumin (RSA), the most abundant protein with the function of transporting endo/exogenous molecules. In this sense, the present study elucidated the mechanism of interaction between PPL and RSA, using in conjunction spectroscopic and computational techniques. This paper shows the importance of applying inner filter correction over the entire fluorescence spectrum prior to any conclusion regarding changes in the polarity of the fluorophore microenvironment, also demonstrates the convergence of the results obtained from the treatment of fluorescence data using the area below the spectrum curve and the intensity in a single wavelength. Thermodynamic parameters revealed that PPL binds to RSA spontaneously (ΔG < 0) and the process is entropically driven. Interaction density function method (IDF) indicated that PPL accessed two cooperative sites in RSA, with moderate binding constants (2.3 × 105 M-1 and 1.3 × 105 M-1). The molecular docking described the microenvironment of the interaction sites, rich in apolar residues. The stability of the RSA-PPL complex was checked by molecular dynamics.