In-depth investigation of the effect of pH on the autofluorescence properties of DPF3b and DPF3a amyloid fibrils.

Double PHD fingers 3 (DPF3) protein exists as two splicing variants, DPF3b and DPF3a, the involvement of which in human cancer and neurodegeneration is beginning to be increasingly recognised. Both isoforms have recently been identified as intrinsically disordered proteins able to undergo amyloid fibrillation. Upon their aggregation, DPF3 proteins exhibit an intrinsic fluorescence in the visible range, referred to as deep-blue autofluorescence (dbAF). Comprehension of such phenomenon remaining elusive, we investigated in the present study the influence of pH on the optical properties of DPF3b and DPF3a fibrils. By varying the excitation wavelength and the pH condition, the two isoforms were revealed to display several autofluorescence modes that were defined as violet, deep-blue, and blue-green according to their emission range. Complementarily, analysis of excitation spectra and red edge shift plots allowed to better decipher their photoselection mechanism and to highlight isoform-specific excitation-emission features. Furthermore, the observed violation to Kasha-Vavilov's rule was attributed to red edge excitation shift effects, which were impacted by pH-mediated H-bond disruption, leading to changes in intramolecular charge and proton transfer, or π-electrons delocalisation. Finally, emergence of different autofluorescence emitters was likely related to structurally distinct fibrillar assemblies between isoforms, as well as to discrepancies in the amino acid composition of their aggregation prone regions.

A cytoplasmic chemoreceptor and reactive oxygen species mediate bacterial chemotaxis to copper.

Chemotaxis is a widespread strategy used by unicellular and multicellular living organisms to maintain their fitness in stressful environments. We previously showed that bacteria can trigger a negative chemotactic response to a copper (Cu)-rich environment. Cu ion toxicity on bacterial cell physiology has been mainly linked to mismetallation events and reactive oxygen species (ROS) production, although the precise role of Cu-generated ROS remains largely debated. Here, using inductively coupled plasma optical emission spectrometry on cell fractionates, we found that the cytoplasmic Cu ion content mirrors variations of the extracellular Cu ion concentration. ROS-sensitive fluorescent probe and biosensor allowed us to show that the increase of cytoplasmic Cu ion content triggers a dose-dependent oxidative stress, which can be abrogated by superoxide dismutase and catalase overexpression. The inhibition of ROS production in the cytoplasm not only improves bacterial growth but also impedes Cu chemotaxis, indicating that ROS derived from cytoplasmic Cu ions mediate the control of bacterial chemotaxis to Cu. We also identified the Cu chemoreceptor McpR, which binds Cu ions with low affinity, suggesting a labile interaction. In addition, we demonstrate that the cysteine 75 and histidine 99 within the McpR sensor domain are key residues in Cu chemotaxis and Cu coordination. Finally, we discovered that in vitro both Cu(I) and Cu(II) ions modulate McpR conformation in a distinct manner. Overall, our study provides mechanistic insights on a redox-based control of Cu chemotaxis, indicating that the cellular redox status can play a key role in bacterial chemotaxis.

Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a.

Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically disordered proteins. DPF3 isoforms differ from their C-terminal region (C-TERb and C-TERa), containing zinc fingers and disordered domains. Herein, we investigated the disorder aggregation properties of C-TER isoforms. In agreement with the predictions, spectroscopy highlighted a lack of a highly ordered structure, especially for C-TERa. Over a few days, both C-TERs were shown to spontaneously assemble into similar antiparallel and parallel ß-sheet-rich fibrils. Altered metal homeostasis being a neurodegeneration hallmark, we also assessed the influence of divalent metal cations, namely Cu2+, Mg2+, Ni2+, and Zn2+, on the C-TER aggregation pathway. Circular dichroism revealed that metal binding does not impair the formation of ß-sheets, though metal-specific tertiary structure modifications were observed. Through intrinsic and extrinsic fluorescence, we found that metal cations differently affect C-TERb and C-TERa. Cu2+ and Ni2+ have a strong inhibitory effect on the aggregation of both isoforms, whereas Mg2+ impedes C-TERb fibrillation and, on the contrary, enhances that of C-TERa. Upon Zn2+ binding, C-TERb aggregation is also hindered, and the amyloid autofluorescence of C-TERa is remarkably red-shifted. Using electron microscopy, we confirmed that the metal-induced spectral changes are related to the morphological diversity of the aggregates. While metal-treated C-TERb formed breakable and fragmented filaments, C-TERa fibrils retained their flexibility and packing properties in the presence of Mg2+ and Zn2+ cations.

Self-standing, conducting and capacitive biomimetic hybrid nanomembranes for selective molecular ion separation.

Hybrid free-standing biomimetic materials are developed by integrating the VDAC36 ß-barrel protein into robust and flexible three-layered polymer nanomembranes. The first and third layers are prepared by spin-coating a mixture of poly(lactic acid) (PLA) and poly(vinyl alcohol) (PVA). PVA nanofeatures are transformed into controlled nanoperforations by solvent-etching. The two nanoperforated PLA layers are separated by an electroactive layer, which is successfully electropolymerized by introducing a conducting sacrificial substrate under the first PLA nanosheet. Finally, the nanomaterial is consolidated by immobilizing the VDAC36 protein, active as an ion channel, into the nanoperforations of the upper layer. The integration of the protein causes a significant reduction of the material resistance, which decreases from 21.9 to 3.9 kΩ cm2. Electrochemical impedance spectroscopy studies using inorganic ions and molecular metabolites (i.e.l-lysine and ATP) not only reveal that the hybrid films behave as electrochemical supercapacitors but also indicate the most appropriate conditions to obtain selective responses against molecular ions as a function of their charge. The combination of polymers and proteins is promising for the development of new devices for engineering, biotechnological and biomedical applications.

Structural and functional characterization of Solanum tuberosum VDAC36.

As it forms water-filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the voltage-dependent anion channel (VDAC) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum, stVDAC36, has been successfully overexpressed and refolded by an in-house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross-linking and molecular modeling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulfide bond between two stVDAC36 monomers. The pore-forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations.

Peptide-surfactant interactions: A combined spectroscopic and molecular dynamics simulation approach.

In the present contribution, we report a combined spectroscopic and computational approach aiming to unravel at atomic resolution the effect of the anionic SDS detergent on the structure of two model peptides, the α-helix TrpCage and the ß-stranded TrpZip. A detailed characterization of the specific amino acids involved is performed. Monomeric (single molecules) and micellar SDS species differently interact with the α-helix and ß-stranded peptides, emphasizing the different mechanisms occurring below and above the critical aggregation concentration (CAC). Below the CAC, the α-helix peptide is fully unfolded, losing its hydrophobic core and its Asp-Arg salt bridge, while the ß-stranded peptide keeps its native structure with its four Trp well oriented. Above the CAC, the SDS micelles have the same effect on both peptides, that is, destabilizing the tertiary structure while keeping their secondary structure. Our studies will be helpful to deepen our understanding of the action of the denaturant SDS on peptides and proteins.

Polypyrrole-supported membrane proteins for bio-inspired ion channels.

Biomedical platforms constructed by immobilizing membrane proteins in matrixes made of synthetic organic polymers is a challenge because the structure and function of these proteins are affected by environmental conditions. In this work, an operative composite that regulates the diffusion of alkali ions has been prepared by functionalizing a supporting matrix made of poly(N-methylpyrrole) (PNMPy) with a ß-barrel membrane protein (Omp2a) that forms channels and pores. The protein has been unequivocally identified in the composite, and its structure has been shown to remain unaltered. The PNMPy-Omp2a platform fulfills properties typically associated with functional bio-interfaces with biomedical applications (e.g., biocompatibility, biodegrabadility, and hydrophilicity). The functionality of the immobilized protein has been examined by studying the passive ion transport response in the presence of electrolytic solutions with Na(+) and K(+) concentrations close to those found in blood. Although the behavior of PNMPy and PNMPy-Omp2a is very similar for solutions with very low concentration, the resistance of the latter decreases drastically when the concentration of ions increases to ∼100 mM. This reduction reflects an enhanced ion exchange between the biocomposite and the electrolytic medium, which is not observed in PNMPy, evidencing that PNMPy-Omp2a is particularly well suited to prepare bioinspired channels and smart biosensors.

The role of 2-methyl-2, 4-pentanediol in sodium dodecyl sulfate micelle dissociation unveiled by dynamic light scattering and molecular dynamics simulations.

The development of efficient protein refolding techniques remains a challenge in biotechnology. In that context, it has recently been reported that the addition of 2-methyl-2, 4-pentanediol (MPD) to sodium dodecyl sulfate (SDS) allows the renaturation of both soluble and membrane proteins. The present work combines experimental (dynamic light scattering; DLS) and theoretical (molecular dynamics) approaches to study the molecular basis of the association between SDS and MPD, in order to understand its relevance in the refolding process. DLS shows the micelle dissociation in the presence of molar concentrations of MPD, and simulations reveal that this process results from a screening of the negative charge on the SDS headgroup and a minimization of the solvent (water) accessibility of the detergent tail. This suggests a mechanism whereby the combination of these effects leads to the shift from a "harsh" to a "gentle" detergent behavior, which in turn promotes a productive refolding of the protein.

An unprecedented reversible mode of action of ß-lactams for the inhibition of human fatty acid amide hydrolase (hFAAH).

A series of compound was prepared to clarify the reversible mechanism of ß-lactamic hFAAH inhibitors on the one hand, and to modulate some of their physicochemical parameters on the other hand. In particular, two compounds (4b and 4e) were designed to display a potential good leaving group on the crucial carbonyl with a view to possibly acylating the active serine of the hFAAH catalytic triad. Reversibility studies showed that these two compounds retain the reversible mode of inhibition, suggesting a noncovalent interaction between our ß-lactams and hFAAH. Finally, pharmacological evaluations of bioisosteres of the lead compound (4a, IC(50) = 5.3 nM) revealed that log P values and PSA could be optimized without altering the FAAH inhibition (IC(50) values from 3.65 nM to 70.9 nM).

Towards a universal method for protein refolding: the trimeric beta barrel membrane Omp2a as a test case.

It has recently been reported that 2-methyl-2,4-pentanediol (MPD) can modulate the protein-binding properties of sodium dodecyl sulfate (SDS), turning it into a non-denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD-based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far-UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4 mg/mL) and ionic strength (up to 1 M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20-40°C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations.

Stochastic simulation of structural properties of natively unfolded and denatured proteins.

A new simulation strategy based on a stochastic process has been developed and tested to study the structural properties of the unfolded state of proteins at the atomistic level. The procedure combines a generation algorithm to produce representative uncorrelated atomistic microstructures and an original relaxation method to minimize repulsive non-bonded interactions. Using this methodology, a set of 14 unfolded proteins, including seven natively unfolded proteins as well as seven "classical" proteins experimentally described in denaturation conditions, has been investigated. Comparisons between the calculated and available experimental values of several properties, at hydrodynamic and atomic level, used to describe the unfolded state, such as the radius of gyration, the maximum length, the hydrodynamic radius, the diffusion coefficient, the sedimentation coefficient, and the NMR chemical shifts, reflect a very good agreement. Furthermore, our results indicate that the relationship between the radius of gyration and the hydrodynamic radius deviates from the Zimm's theory of polymer dynamics for random coils, as was recently observed using single-molecule fluorescent methods. Simulations reveal that the interactions between atoms separated by three chemical bonds (1-4 interactions) play a crucial role in the generation process, suggesting that the unfolded state is essentially governed by bonding and short-range non-bonding interactions.

Synthesis, biological evaluation and molecular modeling studies of quinolonyl diketo acid derivatives: new structural insight into the HIV-1 integrase inhibition.

New quinolonyl diketo acid compounds bearing various substituents at position 6 of the quinolone scaffold were designed and synthesized as potential HIV-1 integrase inhibitors. These new compounds were evaluated for their antiviral and anti-integrase activity and showed inhibitory potency similar to that of 6-bromide analog 2. Molecular modeling and docking studies were performed to rationalize these data and to provide a detailed understanding of the mechanism of inhibition for this class of compounds.

Protecting role of cosolvents in protein denaturation by SDS: a structural study.

BACKGROUND: Recently, we reported a unique approach to preserve the activity of some proteins in the presence of the denaturing agent, Sodium Dodecyl Sulfate (SDS). This was made possible by addition of the amphipathic solvent 2,4-Methyl-2-PentaneDiol (MPD), used as protecting but also as refolding agent for these proteins. Although the persistence of the protein activity in the SDS/MPD mixture was clearly established, preservation of their structure was only speculative until now. RESULTS: In this paper, a detailed X-ray study addresses the pending question. Crystals of hen egg-white lysozyme were grown for the first time in the presence of MPD and denaturing concentrations of SDS. Depending on crystallization conditions, tetragonal crystals in complex with either SDS or MPD were collected. The conformation of both structures was very similar to the native lysozyme and the obtained complexes of SDS-lysozyme and MPD-lysozyme give some insights in the interplay of protein-SDS and protein-MPD interactions. CONCLUSION: This study clearly established the preservation of the enzyme structure in a SDS/MPD mixture. It is hypothesized that high concentrations of MPD would change the properties of SDS and lower or avoid interactions between the denaturant and the protein. These structural data therefore support the hypothesis that MPD avoids disruption of the enzyme structure by SDS and can protect proteins from SDS denaturation.

New R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(phenylaminothiocarbonylamino)-2H-1-benzopyrans structurally related to (+/-)-cromakalim as tissue-selective pancreatic beta-cell K(ATP) channel openers.

The present work was aimed at exploring a series of R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(phenylaminothiocarbonylamino)-2H-1-benzopyrans structurally related to (+/-)-cromakalim and differently substituted at the 4- and 6-positions. The biological effects of these putative activators of ATP-sensitive potassium channels (K(ATP)) were characterized in vitro on the pancreatic endocrine tissue (inhibition of insulin release) and on the vascular smooth muscle tissue (relaxation of aorta rings). The biological activity of these new dimethylchroman derivatives was further compared to that of (+/-)-cromakalim, (+/-)-pinacidil, diazoxide and BPDZ 73. Structure-activity relationships indicated that an improved potency for the pancreatic tissue was obtained by introducing a meta- or a para-electron-withdrawing group such as a chlorine atom on the C-4 phenyl ring, independently of the nature of the halogen atom at the 6-position of the benzopyran nucleus. Most original dimethylchroman thioureas were more potent than their 'urea' homologues and even more potent than diazoxide at inhibiting insulin release. Moreover, and unlike (+/-)-cromakalim or (+/-)-pinacidil, such compounds appeared to be highly selective towards the pancreatic tissue. Radioisotopic and fluorimetric investigations indicated that the new drugs activated pancreatic K(ATP) channels. Lastly, conformational studies suggested that the urea/thiourea dimethylchromans can be regarded as hybrid compounds between cromakalim and pinacidil.

Refolding SDS-denatured proteins by the addition of amphipathic cosolvents.

Sodium dodecyl sulfate (SDS) is a highly effective and widely used protein denaturant. We show that certain amphipathic cosolvents such as 2-methyl-2,4-pentanediol (MPD) can protect proteins from SDS denaturation, and in several cases can refold proteins from the SDS-denatured state. This cosolvent effect is observed with integral membrane proteins and soluble proteins from either the alpha-helical or the beta-sheet structural classes. The SDS/MPD system can be used to study processes involving native protein states, and we demonstrate the reversible thermal denaturation of the outer membrane protein PagP in an SDS/MPD buffer. MPD and related cosolvents can modulate the denaturing properties of SDS, and we describe a simple and effective method to recover refolded, active protein from the SDS-denatured state.

Theoretical Investigation of the Geometries and UV-vis Spectra of Poly(l-glutamic acid) Featuring a Photochromic Azobenzene Side Chain.

The geometries and UV-vis spectra of azobenzene dyes grafted as a side chain on poly(l-glutamic acid) have been investigated using a combination of quantum mechanics/molecular mechanics (QM/MM) and time-dependent density functional theory (TD-DFT) methods at the TD-PBE0/6-311+G(d,p)//B3LYP/6-311G(d,p):Amber ff99 level of theory. The influence of the secondary structure of the polypeptide on the electronic properties of both the trans and cis conformations of azobenzene dyes has been studied. It turns out that the grafted dyes exhibit a red-shift of the π → π* absorption energies mainly due to the auxochromic shift induced by the peptidic group used to link the chromophoric unit to the polypeptide and that specific interactions between the glutamic side chain and the azobenzene moiety lead to a large blue-shift of the n → π* transition.

Gauging a hydrocarbon ruler by an intrinsic exciton probe.

The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid:lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane beta-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26-Trp66 phenol-indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton's expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler-exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment.

Synthesis, molecular modelling and enzymatic evaluation of (+/-)3,5-diphenyl-2-thioxoimidazolidin-4-ones as new potential cyclooxygenase inhibitors.

A series of substituted (+/-)3,5-diphenyl-2-thioxoimidazolin-4-ones was synthesized in order to design new type-2 cyclooxygenase (COX-2) inhibitors. This study has led to molecules which completely inhibit human recombinant COX-2 at 50 microM. Molecular modelling highlighted drug interactions with the active site of both cyclooxygenases and suggested modifications to enhance the selectivity of the compounds. In human blood, COX-2 expression was then induced by LPS, and the inhibitory potency of these drugs was disappointing. This weak activity was attributed to a poor aqueous stability of these imidazolidinones substituted by two aryl in position 3 and 5 (15 min < t(1/2) < 130 min). The improvement of the stability of this heterocycle could generate a novel template to treat COX-associated diseases such as arthritis, rheumatoid polyarthritis and cancer.

Synthesis and activity of a new methoxytetrahydropyran derivative as dual cyclooxygenase-2/5-lipoxygenase inhibitor.

Dual COX-2/5-LO inhibitors are described as potential new therapeutic agents for inflammatory diseases. A surprisingly potent effect of a 5-LO pharmacophoric group on the COX-2 inhibition is presented as well as pharmacological in vitro and in vivo results.