Control of gating mode by a single amino acid residue in transmembrane segment IS3 of the N-type Ca2+ channel

N-type Ca2+ channels can be inhibited by neurotransmitter-induced release of G protein βγ subunits. Two isoforms of Cav2.2 α1 subunits of N-type calcium channels from rat brain (Cav2.2a and Cav2.2b; initially termed rbB-I and rbB-II) have different functional properties. Unmodulated Cav2.2b channels are in an easily activated “willing” (W) state with fast activation kinetics and no prepulse facilitation. Activating G proteins shifts Cav2.2b channels to a difficult to activate “reluctant” (R) state with slow activation kinetics; they can be returned to the W state by strong depolarization resulting in prepulse facilitation. This contrasts with Cav2.2a channels, which are tonically in the R state and exhibit strong prepulse facilitation. Activating or inhibiting G proteins has no effect. Thus, the R state of Cav2.2a and its reversal by prepulse facilitation are intrinsic to the channel and independent of G protein modulation. Mutating G177 in segment IS3 of Cav2.2b to E as in Cav2.2a converts Cav2.2b tonically to the R state, insensitive to further G protein modulation. The converse substitution in Cav2.2a, E177G, converts it to the W state and restores G protein modulation. We propose that negatively charged E177 in IS3 interacts with a positive charge in the IS4 voltage sensor when the channel is closed and produces the R state of Cav2.2a by a voltage sensor-trapping mechanism. G protein βγ subunits may produce reluctant channels by a similar molecular mechanism.

Molecular dynamics of a grid-mounted molecular dipolar rotor in a rotating electric field

Classical molecular dynamics is applied to the rotation of a dipolar molecular rotor mounted on a square grid and driven by rotating electric field E(ν) at T ≃ 150 K. The rotor is a complex of Re with two substituted o-phenanthrolines, one positively and one negatively charged, attached to an axial position of Rh\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{4+}}}\end{equation*}\end{document} in a [2]staffanedicarboxylate grid through 2-(3-cyanobicyclo[1.1.1]pent-1-yl)malonic dialdehyde. Four regimes are characterized by a, the average lag per turn: (i) synchronous (a < 1/e) at E(ν) = |E(ν)| > Ec(ν) [Ec(ν) is the critical field strength], (ii) asynchronous (1/e < a < 1) at Ec(ν) > E(ν) > Ebo(ν) > kT/μ, [Ebo(ν) is the break-off field strength], (iii) random driven (a ≃ 1) at Ebo(ν) > E(ν) > kT/μ, and (iv) random thermal (a ≃ 1) at kT/μ > E(ν). A fifth regime, (v) strongly hindered, W > kT, Eμ, (W is the rotational barrier), has not been examined. We find Ebo(ν)/kVcm−1 ≃ (kT/μ)/kVcm−1 + 0.13(ν/GHz)1.9 and Ec(ν)/kVcm−1 ≃ (2.3kT/μ)/kVcm−1 + 0.87(ν/GHz)1.6. For ν > 40 GHz, the rotor behaves as a macroscopic body with a friction constant proportional to frequency, η/eVps ≃ 1.14 ν/THz, and for ν < 20 GHz, it exhibits a uniquely molecular behavior.

Computation of Surface Electrical Potentials of Plant Cell Membranes : Correspondence to Published Zeta Potentials from Diverse Plant Sources

A Gouy-Chapman-Stern model has been developed for the computation of surface electrical potential (ψ0) of plant cell membranes in response to ionic solutes. The present model is a modification of an earlier version developed to compute the sorption of ions by wheat (Triticum aestivum L. cv Scout 66) root plasma membranes. A single set of model parameters generates values for ψ0 that correlate highly with published ζ potentials of protoplasts and plasma membrane vesicles from diverse plant sources. The model assumes ion binding to a negatively charged site (R− = 0.3074 μmol m−2) and to a neutral site (P0 = 2.4 μmol m−2) according to the reactions R− + IΖ ⇌ RIΖ−1 and P0 + IΖ ⇌ PIΖ, where IΖ represents an ion of charge Ζ. Binding constants for the negative site are 21,500 m−1 for H+, 20,000 m−1 for Al3+, 2,200 m−1 for La3+, 30 m−1 for Ca2+ and Mg2+, and 1 m−1 for Na+ and K+. Binding constants for the neutral site are 1/180 the value for binding to the negative site. Ion activities at the membrane surface, computed on the basis of ψ0, appear to determine many aspects of plant-mineral interactions, including mineral nutrition and the induction and alleviation of mineral toxicities, according to previous and ongoing studies. A computer program with instructions for the computation of ψ0, ion binding, ion concentrations, and ion activities at membrane surfaces may be requested from the authors.

Critical contribution of beta chain residue 57 in peptide binding ability of both HLA-DR and -DQ molecules.

Position 57 in the beta chain of HLA class II molecules maintains an Asp/non-Asp dimorphism that has been conserved through evolution and is implicated in susceptibility to some autoimmune diseases. The latter effect may be due to the influence of this residue on the ability of class II alleles to bind specific pathogenic peptides. We utilized highly homologous pairs of both DR and DQ alleles that varied at residue 57 to investigate the impact of this dimorphism on binding of model peptides. Using a direct binding assay of biotinylated peptides on whole cells expressing the desired alleles, we report several peptides that bind differentially to the allele pairs depending on the presence or absence of Asp at position 57. Peptides with negatively charged residues at anchor position 9 bind well to alleles not containing Asp at position 57 in the beta chain but cannot bind well to homologous Asp-positive alleles. By changing the peptides at the single residue predicted to interact with this position 57, we demonstrate a drastically altered or reversed pattern of binding. Ala analog peptides confirm these interactions and identify a limited set of interaction sites between the bound peptides and the class II molecules. Clarification of the impact of specific class II polymorphisms on generating unique allele-specific peptide binding "repertoires" will aid in our understanding of the development of specific immune responses and HLA-associated diseases.

The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface.

The immunity protein of colicin E7 (ImmE7) can bind specifically to the DNase-type colicin E7 and inhibit its bactericidal activity. Here we report the 1.8-angstrom crystal structure of the ImmE7 protein. This is the first x-ray structure determined in the superfamily of colicin immunity proteins. The ImmE7 protein consists of four antiparallel alpha-helices, folded in a topology similar to the architecture of a four-helix bundle structure. A region rich in acidic residues is identified. This negatively charged area has the greatest variability within the family of DNase-type immunity proteins; thus, it seems likely that this area is involved in specific binding to colicin. Based on structural, genetic, and kinetic data, we suggest that all the DNase-type immunity proteins, as well as colicins, share a "homologous-structural framework" and that specific interaction between a colicin and its cognate immunity protein relies upon how well these two proteins' charged residues match on the interaction surface, thus leading to specific immunity of the colicin.

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Transport of cytoskeletal elements in the squid giant axon.

In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

Synthesis of a thymidyl pentamer of deoxyribonucleic guanidine and binding studies with DNA homopolynucleotides.

Replacement of the phosphodiester linkages of the polyanion DNA with guanidine linkers provides the polycation deoxynucleic guanidine (DNG). The synthesis of pentameric thymidyl DNA is provided. This polycationic DNG species binds with unprecedented affinity and with base-pair specificity to negatively charged poly(dA) to provide both double and triple helices. The dramatic stability of these hybrid structures is shown by their denaturation temperatures (Tm). For example, the double helix of the pentameric thymidyl DNG and poly(dA) does not dissociate in boiling water (ionic strength = 0.12), whereas the Tm for pentameric thymidyl DNA associated with poly(dA) is approximately 13 degrees C (ionic strength = 0.12). The effect of ionic strength on Tm for DNG complexes with DNA shows an opposite correlation compared with double-stranded DNA and is much more dramatic than for double-stranded DNA.

Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase.

It was previously proposed that the activation of rat liver phenylalanine hydroxylase (EC 1.14.16.1) by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16 is due to the introduction of the negatively charged phosphate group. To explore the validity of this proposal, we have applied site-directed mutagenesis to specifically replace Ser-16 with negatively charged amino acids, glutamic and aspartic; with polar uncharged amino acids, asparagine and glutamine; with the positively charged amino acid lysine; and with the nonpolar hydrophobic amino acid alanine. The wild-type and mutant enzymes were purified to homogeneity, and the importance of Ser-16 in the activation of phenylalanine hydroxylase was examined by comparing the state of activation of the phosphorylated form of the wild-type hydroxylase with that of the mutants. The kinetic studies carried out on the wild-type phosphorylated hydroxylase showed that all the activation could be accounted for by an increase in Vmax with no change in Km for either phenylalanine or the pterin cofactor. Replacement of Ser-16 with a negatively charged residue, glutamate of aspartate, resulted in the activation of the hydroxylase by 2- to 4-fold, whereas replacement with glutamine, asparagine, lysine, or alanine resulted in a much more modest increase. Further, lysolecithin was found to stimulate the phosphorylated hydroxylase and the mutant enzymes S16E and S16D by a factor of 6-7. In contrast, the mutants S16Q, S16N, and S16A all showed the same magnitude of activation as the wild-type with lysolecithin. Therefore, this study demonstrates that activation of the enzyme by phosphorylation of Ser-16 by cAMP-dependent protein kinase is due to the introduction of negative charge(s) and strongly suggests the involvement of electrostatic interaction between the regulatory and catalytic domains of the hydroxylase.

The effect of protein concentration on ion binding.

The concentration of protein in a solution has been found to have a significant effect on ion binding affinity. It is well known that an increase in ionic strength of the solvent medium by addition of salt modulates the ion-binding affinity of a charged protein due to electrostatic screening. In recent Monte Carlo simulations, a similar screening has been detected to arise from an increase in the concentration of the protein itself. Experimental results are presented here that verify the theoretical predictions; high concentrations of the negatively charged proteins calbindin D9k and calmodulin are found to reduce their affinity for divalent cations. The Ca(2+)-binding constant of the C-terminal site in the Asn-56 --> Ala mutant of calbindin D9k has been measured at seven different protein concentrations ranging from 27 microM to 7.35 mM by using 1H NMR. A 94% reduction in affinity is observed when going from the lowest to the highest protein concentration. For calmodulin, we have measured the average Mg(2+)-binding constant of sites I and II at 0.325, 1.08, and 3.25 mM protein and find a 13-fold difference between the two extremes. Monte Carlo calculations have been performed for the two cases described above to provide a direct comparison of the experimental and simulated effects of protein concentration on metal ion affinities. The overall agreement between theory and experiment is good. The results have important implications for all biological systems involving interactions between charged species.

Single-exciton spectroscopy of single Mn doped InAs quantum dots

The optical spectroscopy of a single InAs quantum dot doped with a single Mn atom is studied using a model Hamiltonian that includes the exchange interactions between the spins of the quantum dot electron-hole pair, the Mn atom, and the acceptor hole. Our model permits linking the photoluminescence spectra to the Mn spin states after photon emission. We focus on the relation between the charge state of the Mn, A0 or A−, and the different spectra which result through either band-to-band or band-to-acceptor transitions. We consider both neutral and negatively charged dots. Our model is able to account for recent experimental results on single Mn doped InAs photoluminescence spectra and can be used to account for future experiments in GaAs quantum dots. Similarities and differences with the case of single Mn doped CdTe quantum dots are discussed.

Polyaniline/Montmorillonite Nanocomposites Obtained by In Situ Intercalation and Oxidative Polymerization in Cationic Modified-Clay (Sodium, Copper and Iron)

Polyaniline/montmorillonite nanocomposites (PANI/M) were obtained by intercalation of aniline monomer into M modified with different cations and subsequent oxidative polymerization of the aniline. The modified-clay was prepared by ion exchange of sodium, copper and iron cations in the clay (Na–M, Cu–M and Fe–M respectively). Infrared spectroscopy confirms the electrostatic interaction between the oxidized PANI and the negatively charged surface of the clay. X-ray diffraction analysis provides structural information of the prepared materials. The nanocomposites were characterized by transmission electron microscopy and their thermal degradation was investigated by thermogravimetric analysis. The weight loss suggests that the PANI chains in the nanocomposites have higher thermal stability than pure PANI. The electrical conductivity of the nanocomposites increased between 12 and 24 times with respect to the pure M and this increase was dependent on the cation-modification. The electrochemical behavior of the polymers extracted from the nanocomposites was studied by cyclic voltammetry and a good electrochemical response was observed.

Conformational changes of dissolved humic and fulvic superstructures with progressive iron complexation

Conformational changes of a humic acid (HA) and a fulvic acid (FA) induced by iron complexation were followed by high-performance size exclusion chromatography (HPSEC) with both UV–vis and refractive index (RI) detectors. Molecular size distribution was reduced for HA and increased for FA with progressive iron complexation. Since interactions of Fe with humic components are electrostatic, it is likely that the triple-charged Fe ions formed stronger complexes with the more acidic hydrophilic and hydrated FA than with the less acidic and more hydrophobic HA. The large content of ionized carboxyl groups in FA, thus favored the formation of intra- or intermolecular bridges between the negatively charged fulvic acid molecules, and led to more compact and larger size network than for HA. Conversely, iron complexation with HA disrupted the humic conformational arrangements stabilized by only weak hydrophobic bonds into smaller-size aggregates of greater conformational stability due to formation of strong metal complexes. These results confirmed that humic molecules in solution were organized in supramolecular associations of relatively small molecules loosely bound together by dispersive interactions and hydrogen bonds, and they specifically responded to chemical changes brought about by metal additions. The present study revealed the molecular changes occurring in superstructures of natural organic matter when in metal complexes and contributed to understand and predict the environmental behavior in waters and soil of metal complexes with natural organic matter.

Electrochemical Behaviour of PSS-Functionalized Silica Films Prepared by Electroassisted Deposition of Sol–Gel Precursors

Porous, electrically insulating SiO2 layers containing polystyrene sulfonate (PSS) were deposited on glassy carbon electrodes by an electrochemically assisted deposition method. The obtained material was characterized by microscopic, spectroscopic and thermal techniques. Silica-PSS films modify the electrochemical response of the glassy carbon electrodes against selected redox probes. Positively charged species show reduced diffusivities across the SiO2-PSS pores, which resulted in a concentration ratio higher than 1 for these species. The opposite behaviour was found for negatively charged redox probes. These observations can be interpreted in terms of the different affinity of the GC/SiO2-PSS-modified electrode for the electroactive species, as a consequence of the negatively charged porous silica.

Développement d’une lentille cornéenne médicamentée

L’utilisation de lentilles cornéennes peut servir à améliorer le profil d’administration d’un principe actif dans les yeux. Avec une efficacité d’administration de 5% par l’utilisation de gouttes, on comprend rapidement que l’administration oculaire doit être améliorée. Cette faible administration a donné naissance à plusieurs tentatives visant à fabriquer des lentilles cornéennes médicamentées. Cependant, à cause de multiples raisons, aucune de ces tentatives n’a actuellement été mise sur le marché. Nous proposons dans cette étude, une possible amélioration des systèmes établis par le développement d’une lentille cornéenne à base de 2-(hydroxyéthyle)méthacrylate (HEMA), dans laquelle des microgels, à base de poly N-isopropylacrylamide (pNIPAM) thermosensible encapsulant un principe actif, seront incorporé. Nous avons donc débuté par développer une méthode analytique sensible par HPCL-MS/MS capable de quantifier plusieurs molécules à la fois. La méthode résultante a été validée selon les différents critères de la FDA et l’ICH en démontrant des limites de quantifications et de détections suffisamment basses, autant dans des fluides simulés que dans les tissus d’yeux de lapins. La méthode a été validée pour sept médicaments ophtalmiques : Pilocarpine, lidocaïne, proparacaïne, atropine, acétonide de triamcinolone, timolol et prednisolone. Nous avons ensuite fait la synthèse des microgels chargés négativement à base de NIPAM et d’acide méthacrylique (MAA). Nous avons encapsulé une molécule modèle dans des particules ayant une taille entre 200 et 600 nm dépendant de la composition ainsi qu’un potentiel zêta variant en fonction de la température. L’encapsulation de la rhodamine 6G (R6G) dans les microgels a été possible jusqu’à un chargement (DL%) de 38%. L’utilisation des isothermes de Langmuir a permis de montrer que l’encapsulation était principalement le résultat d’interactions électrostatiques entre les MAA et la R6G. Des cinétiques de libérations ont été effectuées à partir d’hydrogels d’acrylamide chargés en microgels encapsulant la R6G. Il a été trouvé que la libération des hydrogels chargés en microgels s’effectuait majoritairement selon l’affinité au microgel et sur une période d’environ 4-24 heures. La libération à partir de ces systèmes a été comparée à des formules d’hydrogels contenant des liposomes ou des nanogels de chitosan. Ces trois derniers (liposomes, microgels et nanogels) ont présenté des résultats prometteurs pour différentes applications avec différents profils de libérations. Enfin, nous avons transposé le modèle développé avec les gels d’acrylamide pour fabriquer des lentilles de contact de 260 à 340 µm d’épaisseur à base de pHEMA contenant les microgels avec une molécule encapsulée devant être administrée dans les yeux. Nous avons modifié la composition de l’hydrogel en incorporant un polymère linéaire, la polyvinylpyrrolidone (PVP). L’obtention d’hydrogels partiellement interpénétrés améliore la rétention d’eau dans les lentilles cornéennes. L’encapsulation dans les microgels chargés négativement a donné de meilleurs rendements avec la lidocaïne et cette dernière a été libérée de la lentille de pHEMA en totalité en approximativement 2 heures qu’elle soit ou non encapsulée dans des microgels. Ainsi dans cette étude pilote, l’impact des microgels n’a pas pu être déterminé et, de ce fait, nécessitera des études approfondies sur la structure et les propriétés de la lentille qui a été développée. En utilisant des modèles de libération plus représentatifs de la physiologie de l’œil, nous pourrions conclure avec plus de certitude concernant l’efficacité d’un tel système d’administration et s’il est possible de l’optimiser.