Physiological Concentrations Of NaCl Research Articles

Chitosan polyplexes: Energetics of formation and conformational changes in DNA upon binding and release

Energetics of chitosan (CS) polyplexes and conformational stability of bound DNA were studied at pH 5.0 by ITC and HS–DSC, respectively. The CS–DNA binding isotherm was well approximated by the McGhee–von Hippel model suggesting the binding mechanism to be a cooperative attachment of interacting CS ligands to the DNA matrix. Melting thermograms of polyplexes revealed the transformation of different conformational forms of bound DNA in dependence on the CS/DNA weight ratio rw. At 0<rw<0.7 two conformational forms were observed and assigned, respectively, to the intact (free) and globular (bound) DNA. A contribution of the globular DNA to the melting enthalpy approached 100 % at the equivalent weight CS content. At higher CS contents a further stabilization of the globular DNA conformation was detected and assigned to the DNA globules in overcharged polyplexes. The polyplex dissociation was studied at pH 7.4 under conditions of zero and a physiological concentration of NaCl. Phase separation of the system was observed in both cases. DNA was almost completely immobilized in the lower phase enriched with CS. At physiological ionic strength, DNA converted from the globular to the intact form although remained to be immobilized in the CS phase.

DNA binding by the antimalarial compound artemisinin

Artemisinin (ART) is a vital medicinal compound that is used alone or as part of a combination therapy against malaria. ART is thought to function by attaching to heme covalently and alkylating a range of proteins. Using a combination of biophysical methods, we demonstrate that ART is bound by three-way junction and duplex containing DNA molecules. Binding of ART by DNA is first shown for the cocaine-binding DNA aptamer and extensively studied using this DNA molecule. Isothermal titration calorimetry methods show that the binding of ART is both entropically and enthalpically driven at physiological NaCl concentration. Native mass spectrometry methods confirm DNA binding and show that a non-covalent complex is formed. Nuclear magnetic resonance spectroscopy shows that ART binds at the three-way junction of the cocaine-binding aptamer, and that binding results in the folding of the structure-switching variant of this aptamer. This structure-switching ability was exploited using the photochrome aptamer switch assay to demonstrate that ART can be detected using this biosensing assay. This study is the first to demonstrate the DNA binding ability of ART and should lay the foundation for further work to study implications of DNA binding for the antimalarial activity of ART.

NaCl increases the dielectric constant of nanoconfined water in phospholipid multilamellar vesicle by enhancing intermolecular orientation correlation rather than rotational freedom of individual molecules

We investigate how the NaCl affects the dielectric constant of nanoconfined water in phospholipid multilamellar vesicles using dielectric relaxation spectroscopy. An anomalous increase in the dielectric constant is observed near the physiological NaCl concentration (0<c<0.3M), which has been consistently proposed in molecular dynamic simulations but is first confirmed by experiment. NaCl accelerates the reorientation motion of surface-bound water, which implies an increase of the rotational degree of freedom. However, for water beyond the hydration layer, retarded collective reorientation motion and increased dielectric strength are observed, suggesting NaCl enhances intermolecular orientation correlation rather than rotational freedom.

Supramolecular hydrogelation via host-guest anion recognition: Lamellar hydrogel materials for the release of cationic cargo

Supramolecular hydrogelation via host-guest anion recognition: Lamellar hydrogel materials for the release of cationic cargo

Nanoparticle albumin-bound mTHPC for photodynamic therapy: Preparation and comprehensive characterization of a promising drug delivery system.

Nanoparticle albumin-bound (nab)-technology is an industrial applicable manufacturing method for the preparation of albumin-based drug carriers of poorly water-soluble drugs. In the present study the advantages of nanotechnology, albumin as an endogenous protein with the capability of high tumor enrichment and the selective light activation of the photosensitizer Temoporfin (mTHPC) were combined to a new delivery system for oncological use. The herewith provided well-established photodynamic therapy may enable a beneficial alternative for the treatment of solid tumors. In the present study a reproducible method for the preparation of stable mTHPC-albumin nanoparticles via nab-technology was established. The nanoparticles were physicochemically characterized with regard to particle size and size distribution and the impact of this preparation method on nanoparticle as well as mTHPC stability was investigated. Nanoparticles with improved colloidal stability over a broad pH range and in the presence of physiological NaCl concentrations were achieved in high yield. Due to high pressure homogenization a certain oxidative decay of mTHPC was observed. Cell culture experiments revealed an effective cellular uptake of mTHPC in a cholangiocarcinoma cell line (TFK-1). After light-activation high cytotoxicity was shown for photosensitizer loaded nanoparticles enabling the application of the proposed formulation in photodynamic therapy.

Hydantoin and Its Derivatives Reduce the Viscosity of Concentrated Antibody Formulations by Inhibiting Associations via Hydrophobic Amino Acid Residues

Therapeutic antibodies for subcutaneous (SC) injection must be formulated at high concentrations because of the large therapeutic dose and volume restriction. However, concentrated antibody solutions have undesirably high viscosity, which hampers SC injection. In this study, we demonstrated that hydantoin and its derivatives suppressed the viscosity of concentrated antibody and bovine serum albumin solutions. Hydantoin derivatives, in particular 1-methylhydantoin, appeared more effective. Both hydantoin and 1-methylhydantoin suppressed the viscosity of proteins more effectively when combined with a physiological concentration of NaCl. Moreover, hydantoin rings exhibited thermodynamically favorable interactions with hydrophobic amino acids, as demonstrated using solubility measurements. Molecular dynamics simulations indicated planar stacking interaction or T-shaped interaction between the hydantoin ring structure and the aromatic rings of tryptophan. Thus, the effects of hydantoin compounds in the presence of NaCl on the high viscosity of concentrated protein solutions result from the combined effects between hydantoin and NaCl in suppressing multiple interactions (electrostatic, hydrophobic, π–π, and cation−π interactions) between protein molecules. The obtained data here should be useful for developing therapeutic antibody formulations.

Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state.

Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

Effect of hyaluronic acid on phospholipid model membranes

The role of hyaluronic acid (HA) in supporting low friction and low abrasion during movement in synovial joints is still not fully understood. In this study, we set out to investigate the interaction between HA and representative lipid model membranes, bilayers as well as monolayers, in detail using a variety of calorimetric, spectroscopic, scattering and microscopic techniques, to explore their role in lubrication of articular cartridge. We also cover a wide range of pressures to mimic pressures occurring upon joint movement, aiming at elucidating a possible mechanism for the low friction forces in synovial joints. Effects of HA on lipid bilayer membranes, encompassing significant adsorption at the membrane, penetration of the hydrophobic regions of the HA between lipid head groups, or changes of the temperature- and pressure dependent phase behavior of the membrane or mechanical properties could not be observed. High molecular weight HA at physiological NaCl concentrations might rather operate independently, via an entropy-driven excluded volume effect, to control the hydrodynamics of the synovial fluid. Minor effects are observed only at domain boundaries using lipid monolayers. As lubrication of natural joints is a synergistic effect, other components of the synovial fluid, such as proteoglycans, might play a more active role.

Self-Assembly and Stabilization of Hybrid Cowpea Chlorotic Mottle Virus Particles under Nearly Physiological Conditions.

Capsids of the cowpea chlorotic mottle virus (CCMV) hold great promise for use as nanocarriers in vivo. A major drawback, however, is the lack of stability of the empty wild-type virus particles under physiological conditions. Herein, the assembly behavior and stability under nearly physiological conditions of protein-based block copolymers composed of the CCMV capsid protein and two hydrophobic elastin-like polypeptides are reported. UV/Vis spectroscopy studies, dynamic light-scattering analysis, and TEM measurements demonstrate that both hybrid variants form stable capsids at pH 7.5, physiological NaCl concentration, and 37 °C. The more hydrophobic variant also remains stable in a cell culture medium. These engineered, hybrid CCMV capsid particles can therefore be regarded as suitable candidates for in vivo applications.

Beta-defensin derived cationic antimicrobial peptides with potent killing activity against gram negative and gram positive bacteria

BackgroundAvian β-defensins (AvBD) are cationic antimicrobial peptides (CAMP) with broad-spectrum antimicrobial activity, chemotactic property, and low host cytotoxicity. However, their bactericidal activity is greatly compromised under physiological salt concentrations which limits the use of these peptides as therapeutic agents. The length and the complex structure involving three conserved disulfide bridges are additional drawbacks associated with high production cost. In the present study, short linear CAMPs (11 to 25 a.a. residues) were developed based on the key functional components of AvBDs with additional modifications. Their biological functions were characterized.ResultsCAMP-t1 contained the CCR2 binding domain (N-terminal loop and adjacent α-helix) of AvBD-12 whereas CAMP-t2 comprised the key a.a. residues responsible for the concentrated positive surface charge and hydrophobicity of AvBD-6. Both CAMP-t1 and CAMP-t2 demonstrated strong antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus pseudintermedius. However, CAMP-t1 failed to show chemotactic activity and CAMP-t2, although superior in killing Staphylococcus spp., remained sensitive to salts. Using an integrated design approach, CAMP-t2 was further modified to yield CAMP-A and CAMP-B which possessed the following characteristics: α-helical structure with positively and negatively charged residues aligned on the opposite side of the helix, lack of protease cutting sites, C-terminal poly-Trp tail, N-terminal acetylation, and C-terminal amidation. Both CAMP-A and CAMP-B demonstrated strong antimicrobial activity against multidrug-resistant P. aeruginosa and methicillin-resistant S. pseudintermedius (MRSP) strains. These peptides were resistant to major proteases and fully active at physiological concentrations of NaCl and CaCl2. The peptides were minimally cytotoxic to avian and murine cells and their therapeutic index was moderate (≥ 4.5).ConclusionsAn integrated design approach can be used to develop short and potent antimicrobial peptides, such as CAMP-A and CAMP-B. The advantageous characteristics, including structural simplicity, resistance to salts and proteases, potent antimicrobial activity, rapid membrane attacking mode, and moderate therapeutic index, suggest that CAMP-A and CAMP-B are excellent candidates for development as therapeutic agents against multidrug-resistant P. aeruginosa and methicillin-resistant staphylococci.

Salt Removal from Microliter Sample Volumes by Multiple Phase Microelectromembrane Extractions Across Free Liquid Membranes.

A new concept for rapid and efficient salt removal from minute volumes of saline samples is presented by using multiple phase microelectromembrane extraction (μ-EME). A disposable μ-EME unit is filled with five consecutive plugs of immiscible aqueous and organic solutions; the aqueous sample forms the central phase and is encompassed by two free liquid membranes (FLMs) and two extraction solutions. Salt cations and anions migrate in opposite directions from the sample solution on application of 300 V dc electric potential, cross the neighboring FLM, and are transferred to the corresponding extraction solution. At the same time, the two FLMs selectively eliminate μ-EME transfers of target analytes, which are retained in the sample, and the resulting salt-free sample is then used for subsequent analysis. At optimized conditions (FLMs consisted of 1-pentanol and extraction solutions of deionized water and 100 mM NH4OH), more than 99.3% NaCl was removed from samples containing physiological NaCl concentrations. Simultaneously, more than 95% of model biochemical species (human serum albumin, neurotensin, creatinine, glycine, and alanine) were retained in the samples for subsequent analyses. Good quality capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) electropherograms and interference-free electrospray ionization mass spectrometry (ESI-MS) spectra of selected biochemical species were obtained after μ-EME salt removal from samples containing micromolar concentrations of the target analytes and 150 mM of NaCl.

Crystal Structure of Amylomaltase from Corynebacterium glutamicum.

Amylomaltase is an essential enzyme in maltose utilization and maltodextrin metabolism, and it has been industrially used for the production of cyclodextrin and modification of starch. We determined the crystal structure of amylomaltase from Corynebacterium glutamicum (CgAM) at a resolution of 1.7 Å. Although CgAM forms a dimer without NaCl, it exists as a monomer in physiological concentration of NaCl. CgAM is composed of N- and C-terminal domains, which can be further divided into two and four subdomains, respectively. It exhibits a unique structural feature at the functionally unknown N-domain and also shows two striking differences at the C-domain compared to other amylomaltases. These differences at extended edge of the substrate-binding site might affect substrate specificity for large cyclodextrin formation. The bis-tris methane and sulfate molecules bound at the substrate-binding site of our current structure mimic the binding of the hydroxyl groups of glucose bound at subsites -1 and -2, respectively.

The collagen type I segment long spacing (SLS) and fibrillar forms: Formation by ATP and sulphonated diazo dyes

The collagen type I segment long spacing (SLS) crystallite is a well-ordered rod-like molecular aggregate, ∼300nm in length, which is produced in vitro under mildly acidic conditions (pH 2.5–3.5) in the presence of 1mM ATP. The formation of the SLS crystallite amplifies the inherent linear structural features of individual collagen heterotrimers, due to the punctate linear distribution and summation of the bulkier amino acid side chains along the length of individual collagen heterotrimers. This can be correlated structurally with the 67nm D-banded collagen fibril that is found in vivo, and formed in vitro. Although first described many years ago, the range of conditions required for ATP-induced SLS crystallite formation from acid-soluble collagen have not been explored extensively. Consequently, we have addressed biochemical parameters such as the ATP concentration, pH, speed of formation and stability so as to provide a more complete structural understanding of the SLS crystallite. Treatment of collagen type I with 1mM ATP at neutral and higher pH (6.0–9.0) also induced the formation of D-banded fibrils. Contrary to previous studies, we have shown that the polysulphonated diazo dyes Direct red (Sirius red) and Evans blue, but not Congo red and Methyl blue, can also induce the formation of SLS-like aggregates of collagen, but under markedly different ionic conditions to those employed in the presence of ATP. Specifically, pre-formed D-banded collagen fibrils, prepared in a higher than the usual physiological NaCl concentration (e.g. 500mM NaCl, 20mM Tris-HCl pH7.4 or x3 PBS), readily form SLS aggregates when treated with 0.1mM Direct red and Evans blue, but this did not occur at lower NaCl concentrations. These new data are discussed in relation to the anion (Cl−) and polyanion (phosphate and sulphonate) binding by the collagen heterotrimer and their likely role in collagen fibrillogenesis and SLS formation.

Cell-Envelope Remodeling as a Determinant of Phenotypic Antibacterial Tolerance in Mycobacterium tuberculosis

The mechanisms that lead to phenotypic antibacterial tolerance in bacteria remain poorly understood. We investigate whether changes in NaCl concentration toward physiologically higher values affect antibacterial efficacy against Mycobacterium tuberculosis (Mtb), the causal agent of human tuberculosis. Indeed, multiclass phenotypic antibacterial tolerance is observed during Mtb growth in physiologic saline. This includes changes in sensitivity to ethionamide, ethambutol, d-cycloserine, several aminoglycosides, and quinolones. By employing organism-wide metabolomic and lipidomic approaches combined with phenotypic tests, we identified a time-dependent biphasic adaptive response after exposure of Mtb to physiological levels of NaCl. A first rapid, extensive, and reversible phase was associated with changes in core and amino acid metabolism. In a second phase, Mtb responded with a substantial remodelling of plasma membrane and outer lipid membrane composition. We demonstrate that phenotypic tolerance at physiological concentrations of NaCl is the result of changes in plasma and outer membrane lipid remodeling and not changes in core metabolism. Altogether, these results indicate that physiologic saline-induced antibacterial tolerance is kinetically coupled to cell envelope changes and demonstrate that metabolic changes and growth arrest are not the cause of phenotypic tolerance observed in Mtb exposed to physiologic concentrations of NaCl. Importantly, this work uncovers a role for bacterial cell envelope remodeling in antibacterial tolerance, alongside well-documented allterations in respiration, metabolism, and growth rate.

The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag.

BackgroundThe Gag polyprotein is a multifunctional regulator of retroviral replication and major structural component of immature virions. The nucleic acid chaperone (NAC) activity is considered necessary to retroviral Gag functions, but so far, NAC activity has only been confirmed for HIV-1 and RSV Gag polyproteins. The nucleocapsid (NC) domain of Gag is proposed to be crucial for interactions with nucleic acids and NAC activity. The major function of matrix (MA) domain is targeting and binding of Gag to the plasma membrane but MA can also interact with RNA and influence NAC activity of Gag. Here, we characterize RNA binding properties and NAC activity of HIV-2 MA and Gag, lacking p6 domain (GagΔp6) and discuss potential contribution of NC and MA domains to HIV-2 GagΔp6 functions and interactions with RNA.ResultsWe found that HIV-2 GagΔp6 is a robust nucleic acid chaperone. HIV-2 MA protein promotes nucleic acids aggregation and tRNALys3 annealing in vitro. The NAC activity of HIV-2 NC is affected by salt which is in contrast to HIV-2 GagΔp6 and MA. At a physiological NaCl concentration the tRNALys3 annealing activity of HIV-2 GagΔp6 or MA is higher than HIV-2 NC. The HIV-2 NC and GagΔp6 show strong binding to the packaging signal (Ψ) of HIV-2 RNA and preference for the purine-rich sequences, while MA protein binds mainly to G residues without favouring Ψ RNA. Moreover, HIV-2 GagΔp6 and NC promote HIV-2 RNA dimerization while our data do not support MA domain participation in this process in vitro.ConclusionsWe present that contrary to HIV-1 MA, HIV-2 MA displays NAC activity and we propose that MA domain may enhance the activity of HIV-2 GagΔp6. The role of the MA domain in the NAC activity of Gag may differ significantly between HIV-1 and HIV-2. The HIV-2 NC and MA interactions with RNA are not equivalent. Even though both NC and MA can facilitate tRNALys3 annealing, MA does not participate in RNA dimerization in vitro. Our data on HIV-2 indicate that the role of the MA domain in the NAC activity of Gag differs not only between, but also within, retroviral genera.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0245-1) contains supplementary material, which is available to authorized users.

Involvement of Phenylalanine 297 in the Construction of the Substrate Pocket of Human Aminopeptidase B.

Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes the N-terminal basic amino acids of synthetic and peptide substrates and requires a physiological concentration of NaCl for optimal activity. In this study, we used site-directed mutagenesis and molecular modeling to search for an amino acid residue that is critical for the enzymatic properties of human APB. Substitution of Phe297 with Tyr caused a significant decrease in hydrolytic activity toward synthetic and peptide substrates as well as chloride anion sensitivity. Molecular modeling suggests that Phe297 contributes to the construction of the substrate pocket of APB, which is wide enough to hold a chloride anion and allow the interaction of Gln169 with the N-terminal Arg residue of the substrate through bridging with the chloride anion. These results indicate that Phe297 is crucial for the optimal enzymatic activity and chloride anion sensitivity of APB via formation of the optimal structure of the catalytic pocket.

Small angle X-ray scattering studies of CTNNBL1 dimerization and CTNNBL1/CDC5L complex.

The hPrp19/CDC5L complex is a non-snRNP spliceosome complex that plays a key role in the spliceosome activation during pre-mRNA splicing, and CTNNBL1 and CDC5L are essential components of the complex. In this study, to investigate the oligomeric state of CTNNBL1 in solution, we performed small angle X-ray scattering experiments in various concentrations of NaCl. We observed that CTNNBL1 existed as a dimer in physiological NaCl concentrations. Site-directed mutagenesis experiment of CTNNBL1 confirmed that N-terminal capping region and the first four ARM repeats are important for dimerization of the protein. We also found that the positively-charged NLS3-containing region (residues 197–235) of CDC5L bound to the negatively-charged patch of CTNNBL1 and that the CTNNBL1/CDC5L complex formed a heterotetramer consisting of one CTNNBL1 dimer and one CDC5L dimer. Moreover, reconstruction of 3D models of CTNNBL1/CDC5L complexes containing CTNNBL1 and three different truncated forms of CDC5L showed that the CDC5L141–196 region and the CDC5L236–377 region were positioned at the top of the N-terminal capping region and at the bottom of ARM VII of CTNNBL1, respectively.

Dexamethasone prevents hearing loss by restoring glucocorticoid receptor expression in the guinea pig cochlea.

Dexamethasone is widely used in the treatment of various inner ear diseases. However, knowledge about its direct impact on glucocorticoid receptor (GR) expression is still limited. Prospective animal study in male guinea pigs. A therapeutic concentration of dexamethasone (8 mg/mL) or a physiological concentration of NaCl (0.9% solution) were intratympanically injected into the ears of guinea pigs (n = 10 in each case) 14 hours prior to 90 dB noise exposure (1 hour). Eighteen ears were exposed to noise only. Seven untreated ears were used as controls. Auditory brainstem responses were recorded prior to noise exposure or treatment and 2 hours thereafter. The cochleae were removed from the bullae, transferred to fixative, and embedded in paraffin. GR expression was identified immunohistochemically in the cochlea. Local staining intensities were quantified for seven regions by a computer. Dexamethasone application significantly lowered noise-induced hearing loss. Statistically significant alterations in the average GR expression levels were identified exclusively in the spiral ligament. Comparing GR expression at the level of individual ear, numerous highly significant local associations were identified in the other six cochlear regions. The intratympanic application of dexamethasone is suitable for supporting cochlear homeostasis under stress conditions. The lateral wall, mainly responsible for potassium recycling, seems to be the main target in glucocorticoid therapy.

N-terminal fatty acylation of peptides spanning the cationic C-terminal segment of bovine β-defensin-2 results in salt-resistant antibacterial activity

Peptides spanning the C-terminal segment of bovine-β-defensin-2 (BNBD-2) rich in cationic amino acids, show antimicrobial activity. However, they exhibit considerably reduced activity at physiological concentration of NaCl. In the present study, we have investigated whether N-terminal acylation (acetylation and palmitoylation) of these peptides would result in improved antimicrobial activity. N-terminal palmitoylation though increased hydrophobicity of the peptides, did not enhance antimicrobial potency. However, antibacterial activity of these peptides was not attenuated by NaCl. Biophysical studies on the palmitoylated peptides have indicated that antibacterial activity in the presence of NaCl arises due to the ability of the peptides to interact with membranes more effectively. These peptides showed hemolytic activity which was attenuated considerably in the presence of serum and lipid vesicles. In defensin related peptides, fatty acylation would be a convenient way to generate analogs that are active in the presence of salt.

Unveiling the role of the pesticides paraquat and rotenone on α-synuclein fibrillation in vitro

Epidemiological data have suggested that exposure to environmental toxins might be associated with the etiology of Parkinson's disease (PD). In this context, certain agrochemicals are able to induce Parkinsonism in different animal models via the inhibition of mitochondrial complex I, which leads to an increase in both oxidative stress and the death of nigrostriatal neurons. Additionally, in vitro experiments have indicated that pesticides are capable of accelerating the fibrillation of the presynaptic protein α-synuclein (aS) by binding directly to the protein. However, the molecular details of these interactions are poorly understood. In the present work we demonstrate that paraquat and rotenone, two agrochemicals that lead to a Parkinsonian phenotype in vivo, bind to aS via solvent effects rather than through specific interactions. In fact, these compounds produced no significant effects on aS fibrillation under physiological concentrations of NaCl. NMR data suggest that paraquat interacts with the C-terminal domain of the disordered aS monomer. This interaction was markedly reduced in the presence of NaCl, presumably due to the disruption of electrostatic interactions between the protein and paraquat. Interestingly, the effects produced by short-term incubation of paraquat with aS on the protein conformation resembled those produced by incubating the protein with NaCl alone. Taken together, our data indicate that the effects of these agrochemicals on PD cannot be explained via direct interactions with aS, reinforcing the idea that the role of these compounds in PD is limited to the inhibition of mitochondrial complex I and/or the up-regulation of aS.