Thermodynamics of the physisorption of capping agents on silver nanoparticles.

Nanoscale silver particles have growing applications in biomedical and other technologies due to their unique antibacterial, optical, and electrical properties. The preparation of metal nanoparticles requires the action of a capping agent, such as thiol-containing compounds, to provide colloidal stability, prevent agglomeration, stop uncontrolled growth, and attenuate oxidative damage. However, despite the extensive use of these thiol-based capping agents, the structure of the capping agent layers on the metal surface and the thermodynamics of the formation of these layers remains poorly understood. Here, we leverage molecular dynamics simulations and free energy calculation techniques, to study the behavior of citrate and four thiol-containing capping agents commonly used to protect silver nanoparticles from oxidation. We have studied the single-molecule adsorption of these capping agents to the metal-water interface, their coalescence into clusters, and the formation of complete monolayers covering the metal nanoparticle. At sufficiently high concentrations, we find that allylmercaptan, lipoic acid, and mercaptohexanol spontaneously self-assemble into ordered layers with the thiol group in contact with the metal surface. The high density and ordered structure is presumably responsible for their improved protective characteristics relative to the other compounds studied.

Stabilization of Carbon Nanotubes and Graphene by Tween-80: Mechanistic Insights from Spectroscopic and Simulation Studies.

Polyoxyethylene sorbitan monooleate is commonly used to obtain stable dispersions of nanoparticles (NPs) such as carbon nanotubes (CNTs) and graphene. However, the mechanism underlying dispersion is poorly understood. The present study aimed at investigating the mechanism of stabilization of carbon NPs (CNPs), namely, single-walled CNTs (SWCNTs), multi-walled CNTs (MWCNTs), and graphene, by Tween-80 using attenuated total internal reflection-Fourier transform infrared and nuclear magnetic resonance (NMR) spectroscopy. Molecular dynamics (MD) simulations were performed to identify, at the atomic scale, the significant interactions that underlie the adsorption and the stabilizing effect of Tween-80 on CNPs, in this way corroborating the spectroscopy results. Spectroscopic analysis revealed that the alkyl chain tether to SWCNT, MWCNT, and graphene surface, presumably through π-π interactions between the carbon-carbon double bond in the alkyl chain and the aromatic rings of CNPs. The hydrophilic polyethoxylate chains extend into the aqueous environment and stabilize the suspension by steric hindrance. MD simulations also showed that Tween-80 molecules interact with the CNP surface via the alkyl chain, thus corroborating spectroscopy results. MD simulations additionally revealed that Tween-80 aggregates on the CNP surface shifted from planar to micelle-like with increasing Tween-80 ratios, underscoring concentration-dependent changes in the nature of these interactions.

Structural Insights into the Inhibition Site in the Phosphorylcholine Phosphatase Enzyme of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a highly pathogenic Gram-negative microorganism associated with high mortality levels in burned or immunosuppressed patients or individuals affected by cystic fibrosis. Studies support a colonization mechanism whereby P. aeruginosa can breakdown the host cell membrane phospholipids through the sequential action of two enzymes: (I) hemolytic phospholipase C acting upon phosphatidylcholine or sphingomyelin to produce phosphorylcholine (Pcho) and (II) phosphorylcholine phosphatase (PchP) that hydrolyzes Pcho to generate choline and inorganic phosphate. This coordinated action provides the bacteria with carbon, nitrogen, and inorganic phosphate to support growth. Furthermore, PchP exhibits a distinctive inhibition mechanism by high substrate concentration. Here, we combine kinetic assays and computational approaches such as molecular docking, molecular dynamics, and free-energy calculations to describe the inhibitory site of PchP, which shares specific residues with the enzyme's active site. Our study provides insights into a coupled inhibition mechanism by the substrate, allowing us to postulate that the integrity of the inhibition site is needed to the correct functioning of the active site. Our results allow us to gain a better understanding of PchP function and provide the basis for a rational drug design that might contribute to the treatment of infections caused by this important opportunistic pathogen.

KCTD5, a novel TRPM4-regulatory protein required for cell migration as a new predictor for breast cancer prognosis.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cationic channel that regulates cell migration and contractility. Increased TRPM4 expression has been related to pathologies, in which cytoskeletal rearrangement and cell migration are altered, such as metastatic cancer. Here, we identify the K+ channel tetramerization domain 5 (KCTD5) protein, a putative adaptor of cullin3 E3 ubiquitin ligase, as a novel TRPM4-interacting protein. We demonstrate that KCTD5 is a positive regulator of TRPM4 activity by enhancing its Ca2+ sensitivity. We show that through its effects on TRPM4 that KCTD5 promotes cell migration and contractility. Finally, we observed that both TRPM4 and KCTD5 expression are increased in distinct patterns in different classes of breast cancer tumor samples. Together, these data support that TRPM4 activity can be regulated through expression levels of either TRPM4 or KCTD5, not only contributing to increased understanding of the molecular mechanisms involved on the regulation of these important ion channels, but also providing information that could inform treatments based on targeting these distinct molecules that define TRPM4 activity.

Molecular rotors as reporters for viscosity of solutions of collagen like peptides.

We have studied the suitability of using a molecular rotor-based steady-state fluorometric assay for evaluating changes in both the conformation and the viscosity of collagen-like peptide solutions. Our results indicate that a positive charge incorporated on the hydrophobic tail of the BODIPY molecular rotor favours the dye specificity as a reporter for viscosity of these solutions.

A Structural Model of the Inactivation Gate of Voltage-Activated Potassium Channels.

After opening, the Shaker voltage-gated potassium (KV) channel rapidly inactivates when one of its four N-termini enters and occludes the channel pore. Although it is known that the tip of the N-terminus reaches deep into the central cavity, the conformation adopted by this domain during inactivation and the nature of its interactions with the rest of the channel remain unclear. Here, we use molecular dynamics simulations coupled with electrophysiology experiments to reveal the atomic-scale mechanisms of inactivation. We find that the first six amino acids of the N-terminus spontaneously enter the central cavity in an extended conformation, establishing hydrophobic contacts with residues lining the pore. A second portion of the N-terminus, consisting of a long 24 amino acid α-helix, forms numerous polar contacts with residues in the intracellular entryway of the T1 domain. Double mutant cycle analysis revealed a strong relationship between predicted interatomic distances and empirically observed thermodynamic coupling, establishing a plausible model of the transition of KV channels to the inactivated state.

Study of the affinity between the protein kinase PKA and homoarginine-containing peptides derived from kemptide: Free energy perturbation (FEP) calculations.

Protein kinases (PKs) discriminate between closely related sequences that contain serine, threonine, and/or tyrosine residues. Such specificity is defined by the amino acid sequence surrounding the phosphorylatable residue, so that it is possible to identify an optimal recognition motif (ORM) for each PK. The ORM for the protein kinase A (PKA), a well-known member of the PK family, is the sequence RRX(S/T)X, where arginines at the -3 and -2 positions play a key role with respect to the primed phosphorylation site. In this work, differential affinities of PKA for the peptide substrate Kemptide (LRRASLG) and mutants that substitute the arginine residues by the unnatural peptide homoarginine were evaluated through molecular dynamics (MD) and free energy perturbation (FEP) calculations. The FEP study for the homoarginine mutants required previous elaboration of a CHARMM "arginine to homoarginine" (R2B) hybrid topology file which is available in this manuscript as Supporting Information. Mutants substituting the arginine residues by alanine, lysine, and histidine were also considered in the comparison by using the same protocol. FEP calculations allowed estimating the free energy changes from the free PKA to PKA-substrate complex (ΔΔGE→ES ) when Kemptide structure was mutated. Both ΔΔGS→ES values for homoarginine mutants were predicted with a difference below 1 kcal/mol. In addition, FEP correctly predicted that all the studied mutations decrease the catalytic efficiency of Kemptide for PKA. © 2018 Wiley Periodicals, Inc.

Theoretical rationalisation of the photophysics of a TICT excited state of cinnamoyl-coumarin derivatives in homogeneous and biological membrane models.

A new hybrid cinnamoyl-coumarin probe was synthesised to study the formation and dynamics of a twisted internal charge transfer (TICT) excited state in homogeneous and biological membrane models. This probe showed a large bathochromic shift of the fluorescence band with the solvent polarity, which is associated with the decrease in the fluorescence intensity due to fast non-radiative deactivation pathways, ascribed to TICT excited state formation in polar solvents. The calculated potential energy surfaces using density functional theory (DFT) and time dependent-DFT (TD-DFT) along with the energetic barriers calculated using the ABF methodology established the energy requirements for a rotational twisting of the cinnamoyl-coumarin bond for TICT excited state formation. This strategy has allowed estimating the role of the ground state conformation and excited state distribution that, concomitant with fluorescence lifetime measurements, describes in detail dual fluorescence emission from TICT and ICT excited states. Moreover, the high sensitivity of fluorescence lifetimes of the TICT excited state in liposomes allows us to propose the use of this type of probes as a powerful tool for the study of gel and crystalline liquid phases in lipid membrane models. The development of this new approach will allow rationalizing and understanding the photochemical behavior of fluorescent TICT-based probes in constrained biological environments.

Molecular determinants of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) binding to transient receptor potential V1 (TRPV1) channels.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P2 is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P2 binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P2 interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P2 behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P2 binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P2 in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P2 binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.

New Insights into Peptide-Silver Nanoparticle Interaction: Deciphering the Role of Cysteine and Lysine in the Peptide Sequence.

We studied the interaction of four new pentapeptides with spherical silver nanoparticles. Our findings indicate that the combination of the thiol in Cys and amines in Lys/Arg residues is critical to providing stable protection for the silver surface. Molecular simulation reveals the atomic scale interactions that underlie the observed stabilizing effect of these peptides, while yielding qualitative agreement with experiment for ranking the affinity of the four pentapeptides for the silver surface.

The Dynamic Nonprime Binding of Sampatrilat to the C-Domain of Angiotensin-Converting Enzyme.

Sampatrilat is a vasopeptidase inhibitor that inhibits both angiotensin I-converting enzyme (ACE) and neutral endopeptidase. ACE is a zinc dipeptidyl carboxypeptidase that contains two extracellular domains (nACE and cACE). In this study the molecular basis for the selectivity of sampatrilat for nACE and cACE was investigated. Enzyme inhibition assays were performed to evaluate the in vitro ACE domain selectivity of sampatrilat. The inhibition of the C-domain (Ki = 13.8 nM) by sampatrilat was 12.4-fold more potent than that for the N-domain (171.9 nM), indicating differences in affinities for the respective ACE domain binding sites. Interestingly, replacement of the P2 group of sampatrilat with an aspartate abrogated its C-selectivity and lowered the potency of the inhibitor to activities in the micromolar range. The molecular basis for this selective profile was evaluated using molecular modeling methods. We found that the C-domain selectivity of sampatrilat is due to occupation of the lysine side chain in the S1 and S2 subsites and interactions with Glu748 and Glu1008, respectively. This study provides new insights into ligand interactions with the nonprime binding site that can be exploited for the design of domain-selective ACE inhibitors.

Molecular basis of drug resistance in A/H1N1 virus.

New mutants of human influenza virus (A/H1N1) exhibit resistance to antiviral drugs. The mechanism whereby they develop insensitivity to these medications is, however, not yet completely understood. A crystallographic structure of A/H1N1 neuraminidase has been published recently. Using molecular dynamic simulations, it is now possible to characterize at the atomic level the mechanism that underlies the loss of binding affinity of the drugs. In this study, free-energy perturbation was used to evaluate the relative binding free energies of Tamiflu and Relenza with H274Y, N294S, and Y252H neuraminidase mutants. Our results demonstrate a remarkable correlation between theoretical and experimental data, which quantitatively confirms that the mutants are resistant to Tamiflu but are still strongly inhibited by Relenza. The simulations further reveal the key interactions that govern the affinity of the two drugs for each mutant. This information is envisioned to prove useful for the design of novel neuraminidase inhibitors and for the characterization of new potential mutants.

Self-Organization Dynamics of Collagen-like Peptides Crosslinking Is Driven by Rose-Bengal-Mediated Electrostatic Bridges.

The present work focuses on the computational study of the structural micro-organization of hydrogels based on collagen-like peptides (CLPs) in complex with Rose Bengal (RB). In previous studies, these hydrogels computationally and experimentally demonstrated that when RB was activated by green light, it could generate forms of stable crosslinked structures capable of regenerating biological tissues such as the skin and cornea. Here, we focus on the structural and atomic interactions of two collagen-like peptides (collagen-like peptide I (CLPI), and collagen-like peptide II, (CLPII)) in the presence and absence of RB, highlighting the acquired three-dimensional organization and going deep into the stabilization effect caused by the dye. Our results suggest that the dye could generate a ternary ground-state complex between collagen-like peptide fibers, specifically with positively charged amino acids (Lys in CLPI and Arg in CLPII), thus stabilizing ordered three-dimensional structures. The discoveries generated in this study provide the structural and atomic bases for the subsequent rational development of new synthetic peptides with improved characteristics for applications in the regeneration of biological tissues during photochemical tissue bonding therapies.

Nanoengineered Sprayable Therapy for Treating Myocardial Infarction.

We report the development, as well as the in vitro and in vivo testing, of a sprayable nanotherapeutic that uses surface engineered custom-designed multiarmed peptide grafted nanogold for on-the-spot coating of an infarcted myocardial surface. When applied to mouse hearts, 1 week after infarction, the spray-on treatment resulted in an increase in cardiac function (2.4-fold), muscle contractility, and myocardial electrical conductivity. The applied nanogold remained at the treatment site 28 days postapplication with no off-target organ infiltration. Further, the infarct size in the mice that received treatment was found to be <10% of the total left ventricle area, while the number of blood vessels, prohealing macrophages, and cardiomyocytes increased to levels comparable to that of a healthy animal. Our cumulative data suggest that the therapeutic action of our spray-on nanotherapeutic is highly effective, and in practice, its application is simpler than other regenerative approaches for treating an infarcted heart.

Stereoselective interaction of epimeric naproxen-RGD peptides with human serum albumin.

The dependence of the interaction between human serum albumin (HSA) and two epimeric bioconjugates, which contain (S)- or (R)-naproxen (NPX) bound to a cyclopentapeptide with an arginine-glycine-aspartate sequence (cRGD), on the absolute configuration of the naproxen moiety has been studied using several complementary experiments, such as direct physical separation of the unbound compound, fluorescence quenching of the protein, circular dichroism, and laser flash photolysis. The results were compared with those obtained with model compounds, such as (S)- and (R)-NPX, cRGD, and (S)- and (R)-NPX-NHBu amide analogues. Fluorescence quenching of Trp-214 in HSA by the naproxen compounds (NPXs) revealed lower efficiency for (S)-NPX-RGD in quenching the Trp emission as compared to (R)-NPX-cRGD. Laser flash photolysis data together with the association constants gave information about the distribution of each compound in site I and II, as well as about the lifetime of their triplet excited state within each site of HSA. Furthermore, docking modeling agreed with different modes of binding of the epimeric bioconjugates. Thus, although both bioconjugates bound preferentially to site I, only the NPX moiety of (R)-NPX-cRGD was located within the cavity, explaining its efficiency for Trp-214 fluorescence quenching.

Photophysics and photochemistry of dyes bound to human serum albumin are determined by the dye localization.

The photophysics and photochemistry of rose bengal (RB) and methylene blue (MB) bound to human serum albumin (HSA) have been investigated under a variety of experimental conditions. Distribution of the dyes between the external solvent and the protein has been estimated by physical separation and fluorescence measurements. The main localization of protein-bound dye molecules was estimated by the intrinsic fluorescence quenching, displacement of fluorescent probes bound to specific protein sites, and by docking modelling. All the data indicate that, at low occupation numbers, RB binds strongly to the HSA site I, while MB localizes predominantly in the protein binding site II. This different localization explains the observed differences in the dyes' photochemical behaviour. In particular, the environment provided by site I is less polar and considerably less accessible to oxygen. The localization of RB in site I also leads to an efficient quenching of the intrinsic protein fluorescence (ascribed to the nearby Trp residue) and the generation of intra-protein singlet oxygen, whose behaviour is different to that observed in the external solvent or when it is generated by bound MB.

Antioxidant reactivity toward nitroxide probes anchored into human serum albumin. A new model for studying antioxidant repairing capacity of protein radicals.

A new strategy to evaluate accessibility of antioxidants to radical proteins has been developed using nitroxide prefluorescent probes anchored into human serum albumin (HSA). Binding association constants for the nitroxide probes C(343)T and QT with HSA were 5 x 10(4) and 9 x 10(4)M(-1), respectively. Rate constants for the nitroxide reduction by antioxidants in HSA were determined finding k(HSA)/k(buffer) ratio of 0.8, 1.9, and 0.075 for ascorbic acid, Trolox, and caffeic acid, respectively, for the nitroxide C(343)T reduction.

Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states.

In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Large-scale state-dependent membrane remodeling by a transporter protein.

That channels and transporters can influence the membrane morphology is increasingly recognized. Less appreciated is that the extent and free-energy cost of these deformations likely varies among different functional states of a protein, and thus, that they might contribute significantly to defining its mechanism. We consider the trimeric Na+-aspartate symporter GltPh, a homolog of an important class of neurotransmitter transporters, whose mechanism entails one of the most drastic structural changes known. Molecular simulations indicate that when the protomers become inward-facing, they cause deep, long-ranged, and yet mutually-independent membrane deformations. Using a novel simulation methodology, we estimate that the free-energy cost of this membrane perturbation is in the order of 6-7 kcal/mol per protomer. Compensating free-energy contributions within the protein or its environment must thus stabilize this inward-facing conformation for the transporter to function. We discuss these striking results in the context of existing experimental observations for this and other transporters.