Surfactant-mediated amyloidogenesis behavior of stem bromelain; a biophysical insight.

Neurodegenerative disorders are mainly associated with amyloid fibril formation of different proteins. Stem bromelain (SB), a cysteine protease, is known to exist as a molten globule state at pH 10.0. It passes through the identical surrounding (pH 10.0) in the gut epithelium of intestine upon oral administration. Protein-surfactant complexes are widely employed as drug carriers, so the nature of surfactant toward protein is of great interest. The present work describes the effect of cationic surfactants (CTAB & DTAB) and their hydrophobic behavior toward amyloidogenesis behavior of SB at pH 10.0. Multiple approaches including light scattering, far UV-CD, turbidity measurements, and dye binding assay (ThT, Congo red and ANS) were performed to measure the aggregation propensity of SB. Further, we monitored the hydrodynamic radii of aggregates formed using dynamic light scattering technique. Structure of fibrils was also visualized through fluorescence microscopy as well as TEM. At pH 10.0, low concentration of CTAB (0-200 µM) induced amyloid formation in SB as evident from a prominent increase in turbidity and light scattering, gain in ß-sheet content, and enhanced ThT fluorescence intensity. However, further increase in CTAB concentration suppressed the fibrillation phenomenon. In contrast, DTAB did not induce fibril formation at any concentration used (0-500 µM) due to lower hydrophobicity. Net negative charge developed on protein at high pH (10.0) might have facilitated amyloid formation at low concentration of cationic surfactant (CTAB) due to electrostatic and hydrophobic interactions.

Correction: A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins.

[This corrects the article DOI: 10.1371/journal.pone.0158833.].

A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins.

Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra.

Unraveling Comparative Anti-Amyloidogenic Behavior of Pyrazinamide and D-Cycloserine: A Mechanistic Biophysical Insight.

Amyloid fibril formation by proteins leads to variety of degenerative disorders called amyloidosis. While these disorders are topic of extensive research, effective treatments are still unavailable. Thus in present study, two anti-tuberculosis drugs, i.e., pyrazinamide (PYZ) and D-cycloserine (DCS), also known for treatment for Alzheimer's dementia, were checked for the anti-aggregation and anti-amyloidogenic ability on Aß-42 peptide and hen egg white lysozyme. Results demonstrated that both drugs inhibit the heat induced aggregation; however, PYZ was more potent and decelerated the nucleation phase as observed from various spectroscopic and microscopic techniques. Furthermore, pre-formed amyloid fibrils incubated with these drugs also increased the PC12/SH-SY5Y cell viability as compare to the amyloid fibrils alone; however, the increase was more pronounced for PYZ as confirmed by MTT assay. Additionally, molecular docking study suggested that the greater inhibitory potential of PYZ as compare to DCS may be due to strong binding affinity and more occupancy of hydrophobic patches of HEWL, which is known to form the core of the protein fibrils.

Secondary structural changes in guanidinium hydrochloride denatured mammalian serum albumins and protective effect of small amounts of cationic gemini surfactant pentanediyl-α,ω-bis(cetyldimethylammonium bromide) and methyl-ß-cyclodextrin: A spectroscopic study.

In the present study the cationic gemini surfactant assisted refolding of guanidinium hydrochloride (GdCl) denatured mammalian serum albumins viz. sheep serum albumin (SSA), rat serum albumin (RSA) and porcine serum albumin (PSA) using a combination of cationic gemini surfactants, pentanediyl-α,ω-bis(cetyldimethylammonium bromide) (C16H33(CH3)2N(+)-(CH2)5-N(+)(CH3)2C16H33)⋅2Br(-) designated as G5 and methyl-ß-cyclodextrin in the artificial chaperone assisted two step method, is attempted. The studies were carried out in an aqueous medium (pH 7.4) using dynamic light scattering (DLS), circular dichroism (CD), and fluorescence spectroscopy. A perusal of DLS data indicates that against the native hydrodynamic radius (Rh) of 4.3nm in SSA, 3.9nm in PSA and 3.5nm in RSA, the Rh of the said proteins, when refolding is attempted by simple dilution, increases to 21.7nm, 36.6nm and 37.2nm, respectively. Hydrodynamic radii very near to the native protein, i.e., 4.0nm, 4.1nm and 4.4nm for RSA, PSA and SSA respectively, is obtained on the sequential addition of G5 and methyl-ß-cyclodextrin to the denatured protein. Circular dichroism studies corroborate with the DLS data. The results obtained from the multi-technique approach are ascribed to the presence of two charged head-groups and two hydrocarbon tails in the gemini surfactants resulting in a very strong electrostatic and hydrophobic interactions. Based on the present study it is suggested that the gemini surfactants may be utilized in the protein refolding studies and thus may address one of the most pressing demand of biotechnology industry for the development of efficient and inexpensive folding aides.

Protonation favors aggregation of lysozyme with SDS.

Different proteins have different amino acid sequences as well as conformations, and therefore different propensities to aggregate. Electrostatic interactions have an important role in the aggregation of proteins as revealed by our previous report (J. M. Khan et al., PLoS One, 2012, 7, e29694). In this study, we designed and executed experiments to gain knowledge of the role of charge variations on proteins during the events of protein aggregation with lysozyme as a model protein. To impart positive and negative charges to proteins, we incubated lysozyme at different pH values of below and above the pI (∼11). Negatively charged SDS was used to 'antagonize' positive charges on lysozyme. We examined the effects of pH variations on SDS-induced amyloid fibril formation by lysozyme using methods such as far-UV circular dichroism, Rayleigh scattering, turbidity measurements, dye binding assays and dynamic light scattering. We found that sub-micellar concentrations of SDS (0.1 to 0.6 mM) induced amyloid fibril formation by lysozyme in the pH range of 10.0-1.0 and maximum aggregation was observed at pH 1.0. The morphology of aggregates was fibrillar in structure, as visualized by transmission electron microscopy. Isothermal titration calorimetry studies demonstrated that fibril formation is exothermic. To the best of our current understanding of the mechanism of aggregation, this study demonstrates the crucial role of electrostatic interactions during amyloid fibril formation. The model proposed here will help in designing molecules that can prevent or reverse the amyloid fibril formation or the aggregation.

Nanoparticles in relation to peptide and protein aggregation.

Over the past two decades, there has been considerable research interest in the use of nanoparticles in the study of protein and peptide aggregation, and of amyloid-related diseases. The influence of nanoparticles on amyloid formation yields great interest due to its small size and high surface area-to-volume ratio. Targeting nucleation kinetics by nanoparticles is one of the most searched for ways to control or induce this phenomenon. The observed effect of nanoparticles on the nucleation phase is determined by particle composition, as well as the amount and nature of the particle's surface. Various thermodynamic parameters influence the interaction of proteins and nanoparticles in the solution, and regulate the protein assembly into fibrils, as well as the disaggregation of preformed fibrils. Metals, organic particles, inorganic particles, amino acids, peptides, proteins, and so on are more suitable candidates for nanoparticle formulation. In the present review, we attempt to explore the effects of nanoparticles on protein and peptide fibrillation processes from both perspectives (ie, as inducers and inhibitors on nucleation kinetics and in the disaggregation of preformed fibrils). Their formulation and characterization by different techniques have been also addressed, along with their toxicological effects, both in vivo and in vitro.

Concentration-dependent antagonistic persuasion of SDS and naphthalene derivatives on the fibrillation of stem bromelain.

Sodium dodecyl sulfate, a biological membrane mimetic, can be used to study the conversion of globular proteins into amyloid fibrils in vitro. Using multiple approaches, the effect of SDS was examined on stem bromelain (SB), a widely recognized therapeutic protein. SB is known to exist as a partially folded intermediate at pH 2.0, situation also encountered in the gastrointestinal tract (its site of absorption). In the presence of sub-micellar SDS concentration (500-1000 µM), this intermediate was found to exhibit great propensity to form large-sized ß-sheeted aggregates with fibrillar morphology, the hall marks of amyloid structure. We also observed inhibition of fibrillation by two naphthalene-based compounds, ANS and bis-ANS. While bis-ANS significantly inhibited fibril formation at 50 µM, ANS did so at relatively higher concentration (400 µM). Alcohols, but not salts, were found to weaken the inhibitory action of these compounds suggesting the possible involvement of hydrophobic interactions in their binding to protein. Besides, isothermal titration calorimetry and molecular docking studies suggested that inhibition of fibrillation by these naphthalene derivatives is mediated not just through hydrophobic forces, but also by disruption of π-π interactions between the aromatic residues together with the inter-polypeptide chain repulsion among negatively charged ANS/bis-ANS bound SB.

Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form.

Banana lectin (BL) is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS) binding, size exclusion chromatography (SEC) and dynamic light scattering (DLS). During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml) at pH 2.0 while single peak (61.45 ml) at pH 7.4. The hydrodynamic radii (R h) of native BL was 2.9 nm while at pH 2.0 two species were found with R h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high Cm and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.

Revisiting ligand-induced conformational changes in proteins: essence, advancements, implications and future challenges.

Ligand-induced conformational changes are of immense importance for the biological activity of a protein. An in-depth understanding of salutary and deleterious effects of ligand-induced conformational alterations in single- and multi-chain proteins would lend a hand in human welfare. Unlike single-chain proteins, the function of multichain proteins depends upon the inherent properties of the subunit interfaces. The interfaces of temporary oligomeric proteins and the active sites of enzymes are of similar characteristics but the interfaces are more conservative than the active sites. Therefore, these interfaces may possibly be represented as drug targets by inhibition or induction of the oligomerization process. Thus without detailed structural understanding of ligand-induced conformational changes in a protein, structure-based rational drug designing is a great challenging task. So the purpose of this review is to clarify or enlighten the reader at the degree of internal motions related to protein backbone and side-chain flexibility which occur on binding of small molecule to a protein target. This can prove helpful to improve the conformational prediction for a protein-ligand complex. Besides a detailed description of protein-ligand interaction, this review also focuses on structure-activity relationships of protein which will surely help in the rational drug designing.

1-Anilino-8-naphthalene sulfonate (ANS) is not a desirable probe for determining the molten globule state of chymopapain.

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0-7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.

SDS can be utilized as an amyloid inducer: a case study on diverse proteins.

Sodium dodecyl sulphate (SDS), an anionic surfactant that mimics some characteristics of biological membrane has also been found to induce aggregation in proteins. The present study was carried out on 25 diverse proteins using circular dichroism, fluorescence spectroscopy, dye binding assay and electron microscopy. It was found that an appropriate molar ratio of protein to SDS readily induced amyloid formation in all proteins at a pH below two units of their respective isoelectric points (pI) while no aggregation was observed at a pH above two units of pI. We also observed that electrostatic interactions play a leading role in the induction of amyloid. This study can be used to design or hypothesize a molecule or drug, which may counter act the factor responsible for amyloid formation.