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David S Cafiso - One of the best experts on this subject based on the ideXlab platform.

  • position of synaptotagmin i at the Membrane Interface cooperative interactions of tandem c2 domains
    Biochemistry, 2006
    Co-Authors: Dawn Z Herrick, Stephenie Sterbling, Katie A Rasch, And Anne Hinderliter, David S Cafiso
    Abstract:

    Synaptotagmin I is a synaptic vesicle associated Membrane protein that appears to regulate Ca2+-mediated exocytosis. Here, the Ca2+-dependent Membrane interactions of a water soluble fragment of synaptotagmin I (C2AB) that contains its two C2 domains (C2A and C2B) were determined using site-directed spin labeling. Membrane depth parameters were obtained for 19 spin-labeled mutants of C2AB when bound to phosphatidylcholine and phosphatidylserine Membranes, and these distance constraints were used in combination with the high-resolution structures of C2A and C2B to generate a model for the Membrane orientation and position of synaptotagmin at the bilayer Interface. Both C2A and C2B bind to the Membrane Interface with their first and third Ca2+ binding loops penetrating the Membrane Interface. The polybasic face of C2B does not interact with the Membrane lipid but is available for electrostatic interaction with other components of the fusion machinery. When compared to positions determined previously for the...

  • location of the myristoylated alanine rich c kinase substrate marcks effector domain in negatively charged phospholipid bicelles
    Biophysical Journal, 2003
    Co-Authors: Jeffrey F Ellena, Christine M Burnitz, David S Cafiso
    Abstract:

    The effector domain of the myristoylated alanine-rich C-kinase substrate (MARCKS-ED) is a highly basic, unstructured protein segment that is responsible for attaching MARCKS reversibly to the Membrane Interface. When attached to the Interface, it also has the capacity to sequester phosphoinosities, such as PI(4,5)P2, within the plane of the bilayer. Here, the position of the MARCKS-ED was determined when bound to phospholipid bicelles using high-resolution NMR methods. Two sets of data indicate that the phenylalanine residues of the MARCKS-ED are positioned within the Membrane hydrocarbon a few angstroms from the aqueous-hydrocarbon Interface. First, short-range nuclear Overhauser effects are detected between the aromatic side chains and the lipid acyl chain methylenes. Second, paramagnetic enhancements of nuclear relaxation, produced by molecular oxygen, are similar for the phenylalanine aromatic protons and those observed for protons in the upper portion of the acyl chain. The rates of amide-water proton exchange are fast and only slightly hindered when the peptide is bound to bicelles, indicating that the backbone does not lie within the Membrane hydrocarbon. These results indicate that highly charged peptides such as the MARCKS-ED penetrate the Membrane Interface with aromatic amino acid side chains inserted into the hydrocarbon and the peptide backbone lying within the bilayer Interface. This position may serve to enhance the electrostatic fields produced by this basic domain at the Membrane Interface and may play a role in the ability of the MARCKS-ED to sequester polyphosphoinositides.

  • Membrane bound orientation and position of the synaptotagmin i c2a domain by site directed spin labeling
    Biochemistry, 2003
    Co-Authors: April A Frazier, And Anne Hinderliter, Christina R Roller, Jessica J Havelka, David S Cafiso
    Abstract:

    Site-directed spin labeling was used to determine the Membrane orientation and insertion of the C2A domain from synaptotagmin I. A series of single cysteine mutants of the C2A domain of synaptotagmin I was prepared and labeled with a sulfhydryl specific spin label. Upon Ca2+ or Membrane binding, the EPR line shapes of these mutants reveal dramatic decreases in label mobility within the Ca2+-binding loops. This loss in mobility is likely due in part to a reduction in local backbone fluctuations within the loop regions. Power saturation was then used to determine the position of each spin-labeled site along the bilayer normal, and these EPR distance constraints were used along with the high-resolution solution structure of C2A to generate a model for the orientation and position of the domain at the Membrane Interface. This model places the polypeptide backbone of both the first and third Ca2+-binding loops in contact with the Membrane Interface, with several labeled side chains lying within the bilayer int...

  • Membrane structure of protein kinase c and calmodulin binding domain of myristoylated alanine rich c kinase substrate determined by site directed spin labeling
    Biochemistry, 1996
    Co-Authors: Zhihai Qin, David S Cafiso
    Abstract:

    Cysteine-substituted peptides based on the Membrane, calmodulin, and protein kinase C binding domain of the myristoylated alanine rich C kinase substrate (MARCKS) were synthesized and derivatized with a sulfhydryl reactive proxyl nitroxide. These spin-labeled peptides were used in combination with continuous wave power saturation electron paramagnetic resonance (EPR) spectroscopy to determine the position and structure of the peptide on Membranes containing phosphatidylserine. These peptides bind at the Membrane Interface, with nitroxide side chains in the central and C-terminal regions lying several angstroms below the level of the head group. In contrast, the N-terminus of the peptide is extended out of the Membrane Interface so that the two N-terminal residues are positioned on the aqueous side of the head group. When bound to the Membrane, the N-terminal segment of this peptide is sensitive to the Membrane surface charge density. Higher charge densities decrease the amplitude of side chain motions at the N-terminus and bring this end of the peptide closer to the Membrane Interface. When the location of successive residues along the bilayer normal is compared, no helical trend is seen, and no evidence for aggregation of the peptide is found. The EPR spectra of double spin-labeled peptides also show no evidence for a helical structure. Thus, these basic peptides are in an extended configuration at the Membrane Interface with hydrophobic side chains oriented inward toward the Membrane hydrocarbon.

  • Membrane structure of protein kinase c and calmodulin binding domain of myristoylated alanine rich c kinase substrate determined by site directed spin labeling
    Biochemistry, 1996
    Co-Authors: David S Cafiso
    Abstract:

    Cysteine-substituted peptides based on the Membrane, calmodulin, and protein kinase C binding domain of the myristoylated alanine rich C kinase substrate (MARCKS) were synthesized and derivatized with a sulfhydryl reactive proxyl nitroxide. These spin-labeled peptides were used in combination with continuous wave power saturation electron paramagnetic resonance (EPR) spectroscopy to determine the position and structure of the peptide on Membranes containing phosphatidylserine. These peptides bind at the Membrane Interface, with nitroxide side chains in the central and C-terminal regions lying several angstroms below the level of the head group. In contrast, the N-terminus of the peptide is extended out of the Membrane Interface so that the two N-terminal residues are positioned on the aqueous side of the head group. When bound to the Membrane, the N-terminal segment of this peptide is sensitive to the Membrane surface charge density. Higher charge densities decrease the amplitude of side chain motions at ...

Anthony Kucernak - One of the best experts on this subject based on the ideXlab platform.

  • An anomalous peak observed in the electrochemistry of the platinum/perfluorosulfonic acid Membrane Interface
    2020
    Co-Authors: Alice E.s. Sleightholme, Anthony Kucernak
    Abstract:

    Abstract A solid-state cell is used to study the electrochemistry of platinum at a perfluorosulfonic acid Membrane. An anomalous peak is observed in the platinum electrochemistry at approximately 0.6 0.65 V vs. RHE. The plausible origins of this feature are discussed and experiments which were carried out to characterise the conditions under which the anomalous peak is observed are described. Experiments rule out the possibility of contamination and show that conditions of slow scan rate and low Membrane hydration facilitate the appearance of the peak. Scan rate tests indicate that the anomalous feature owes to a surface process. A possible explanation for the origin of the peak is the formation of oxygenated species on the platinum surface

  • Mass transport and kinetics of electrochemical oxygen reduction at nanostructured platinum electrode and solid polymer electrolyte Membrane Interface
    Journal of Solid State Electrochemistry, 2012
    Co-Authors: Junhua Jiang, Anthony Kucernak
    Abstract:

    Oxygen reduction reaction ( orr ) at nanostructured Pt electrode in a flooded polymer electrolyte Membrane fuel cell environment has been investigated using a nanoporous Pt–Nafion Membrane composite microelectrode by means of steady-state voltammetry and chronoamperometry. The interfacial mass transport of dissolved oxygen is characterized by comparable diffusion coefficients and lower concentrations as compared with literature data obtained with a humidified Membrane. The exchange current densities measured at the nanoporous Pt and Membrane Interface are higher than those reported for the orr in acidic solutions or at polycrystalline Pt and Nafion Membrane Interface, indicating the improvement of the orr kinetics. Increasing temperature substantially improves the orr kinetics and accelerates the diffusion of oxygen, as expected by their Arrhenius behavior. At the nanoporous Pt and Membrane Interface, the Tafel plot exhibits an unusual slope of around 240 mV dec^−1 at high overpotentials. This Tafel slope doubling the value of 120 mV dec^−1 normally reported for the orr in acidic media and at the polycrystalline Pt and Membrane Interface is a signature of non-uniform polarization of the nanoporous Pt electrode on the Membrane which origins have been discussed.

  • oxygen reduction at the silver hydroxide exchange Membrane Interface
    Electrochemistry Communications, 2008
    Co-Authors: Alice E.s. Sleightholme, John R Varcoe, Anthony Kucernak
    Abstract:

    Abstract A solid-state cell is used to study the electrocatalysis of oxygen reduction at the silver/hydroxide-exchange Membrane Interface. The catalyst/Membrane Interface exhibits improved performance in comparison to a catalyst/aqueous sodium hydroxide Interface. Surprisingly, the half-wave potential for oxygen reduction is shown to shift 185 mV higher at the silver/hydroxide-exchange Membrane Interface than for the silver/aqueous hydroxide solution Interface, and the exchange current density is significantly higher at 1.02 × 10 −6  A m −2 . On a cost per performance basis, silver electrocatalysts in a hydroxide-exchange Membrane fuel cell may provide better performance than platinum in a proton-exchange Membrane fuel cell.

Sanjeev Mukerjee - One of the best experts on this subject based on the ideXlab platform.

  • mass transport and oxygen reduction kinetics at an anion exchange Membrane Interface microelectrode studies on effect of carbonate exchange
    ECS Electrochemistry Letters, 2012
    Co-Authors: Iromie Gunasekara, Myoungseok Lee, Daniel F Abbott, Sanjeev Mukerjee
    Abstract:

    Development of proton exchange Membrane fuel cells, have to be tempered with the cost, a significant portion of which is due to the so called ‘stability criterion’ restricting the choice to Pt and Pt alloy materials.Fuelcellsoperating inalkaline media havepotential advantage of facile kinetics of oxygen reduction reaction on non-precious group metals and stability at high pH values. In addition, the hydrodynamics of alkaline Membrane fuel cells is potentially beneficial considering the problems associated with water management in a conventional PEM fuel cell. Further, highly stable PTFE based Membranes are not required under alkaline conditions as the Membranes are less prone to attack by peroxide ions. Even though AEMFCs alleviate most of the hurdles associated with PEM fuel cells, state of art performance of H2/air does not exceed half of the performance shown by H2 fed PEM fuel cells using Pt based catalysts (compare 700 mW cm −2 for PEMFCs 1 at 0.65 V

  • oxygen reduction and transport characteristics at a platinum and alternative proton conducting Membrane Interface
    Journal of Electroanalytical Chemistry, 2004
    Co-Authors: Lei Zhang, Sanjeev Mukerjee
    Abstract:

    Kinetic and mass-transport properties were investigated for the oxygen reduction reaction for Nafion 117 and sulfonated poly(arylene ether sulfone) Membranes, both pre- and post-sulfonated analogs (SPES-40 & SPES-PS) under 100% relative humidity and as a function of pressure (1–4 atm total pressure, 323 K) and temperature (303–353 K, 3 atm) using a solid-state electrochemical cell. Kinetic parameters were obtained using slow-sweep voltammetry while mass-transport parameters, the diffusion coefficient (D) and solubility (C), were obtained using chronoamperometry at a Pt (microelectrode)jproton exchange Membrane (PEM) Interface. The oxygen reduction kinetics were found to be similar for all the Membranes at the Pt microelectrode Interface. The temperature dependence of O2 permeation parameters showed identical trends for the Membranes studied while the pressure dependence of O2 permeation parameters displayed some differences. Despite higher ion exchange capacities and hence higher water uptake, the two SPES Membranes exhibited relatively lower values of D as compared to Nafion 117. The results are discussed in the context of their different microstructures. 2004 Published by Elsevier B.V.

Amitabha Chattopadhyay - One of the best experts on this subject based on the ideXlab platform.

  • Membrane dipole potential is sensitive to cholesterol stereospecificity implications for receptor function
    Chemistry and Physics of Lipids, 2014
    Co-Authors: Suman Bandari, Hirak Chakraborty, Douglas F Covey, Amitabha Chattopadhyay
    Abstract:

    Dipole potential is the potential difference within the Membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the Membrane Interface. Cholesterol, an essential lipid in higher eukaryotic Membranes, has previously been shown to increase Membrane dipole potential. In this work, we explored the effect of stereoisomers of cholesterol, ent-cholesterol and epi-cholesterol, on Membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that cholesterol and ent-cholesterol share comparable ability in increasing Membrane dipole potential. In contrast, epi-cholesterol displays a slight reduction in Membrane dipole potential. Our results constitute the first report on the effect of stereoisomers of cholesterol on Membrane dipole potential, and imply that an extremely subtle change in sterol structure can significantly alter the dipolar field at the Membrane Interface. These results assume relevance in the context of differential abilities of these stereoisomers of cholesterol in supporting the activity of the serotonin1A receptor, a representative G protein-coupled receptor. The close correlation between Membrane dipole potential and receptor activity provides new insight in receptor-cholesterol interaction in terms of stereospecificity. We envision that Membrane dipole potential could prove to be a sensitive indicator of lipid-protein interactions in biological Membranes.

  • influence of ester and ether linkage in phospholipids on the environment and dynamics of the Membrane Interface a wavelength selective fluorescence approach
    Langmuir, 2005
    Co-Authors: Soumi Mukherjee, Amitabha Chattopadhyay
    Abstract:

    We have monitored the environment and dynamics of the Membrane Interface formed by the ester-linked phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the ether-linked phospholipid 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) utilizing the wavelength-selective fluorescence approach and using the fluorescent Membrane probe 2-(9-anthroyloxy)stearic acid (2-AS). This interfacially localized probe offers a number of advantages over those which lack a fixed location in the Membrane. When incorporated in Membranes formed by DPPC and DHPC, 2-AS exhibits red edge excitation shift (REES) of 14 and 8 nm, respectively. This implies that the rate of solvent reorientation, as sensed by the interfacial anthroyloxy probe, in ester-linked DPPC Membranes is slow compared to the rate of solvent reorientation in ether-linked DHPC Membranes. In addition, the fluorescence polarization values of 2-AS are found to be higher in DHPC Membranes than in DPPC Membranes. This is further supported by wavelength-dependent changes in fluorescence polarization and lifetime. Taken together, these results are useful in understanding the role of interfacial chemistry on Membrane physical properties.

Melanie J Cocco - One of the best experts on this subject based on the ideXlab platform.

  • protein folding at the Membrane Interface the structure of nogo 66 requires interactions with a phosphocholine surface
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: S Vasudevan, Chunyi Zhou, Jessica D Schulz, Melanie J Cocco
    Abstract:

    Repair of damage to the central nervous system (CNS) is inhibited by the presence of myelin proteins that prevent axonal regrowth. Consequently, growth inhibitors and their common receptor have been identified as targets in the treatment of injury to the CNS. Here we describe the structure of the extracellular domain of the neurite outgrowth inhibitor (Nogo) in a Membrane-like environment. Isoforms of Nogo are expressed with a common C terminus containing two transMembrane (TM) helices. The ectodomain between the two TM helices, Nogo-66, is active in preventing axonal growth [GrandPre T, Nakamura F, Vartanian T, Strittmatter SM (2000) Nature 403:439-444]. We studied the structure of Nogo-66 alone and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles and dodecylphosphocholine (DPC) micelles as Membrane mimetics. We find that Nogo-66 is largely disordered when free in solution. However, when bound to a phosphocholine surface Nogo-66 adopts a unique, stable fold, even in the absence of TM anchors. Using paramagnetic probes and protein-DPC nuclear Overhauser effects (NOEs), we define portions of the growth inhibitor likely to be accessible on the cell surface. With these data we predict that residues (28-58) are available to bind the Nogo receptor, which is entirely consistent with functional assays. Moreover, the conformations and relative positions of side chains recognized by the receptor are now defined and provide a foundation for antagonist design.

  • protein folding at the Membrane Interface the structure of nogo 66
    Biophysical Journal, 2010
    Co-Authors: Melanie J Cocco, S Vasudevan, Rosemarie Vithayathil, Jessica Schulz, Chunyi Zhou, Sudipta Majumdar, Gregory A Weiss
    Abstract:

    Compelling evidence indicates that repair of damage to the central nervous system (CNS) is inhibited by the presence of protein factors within myelin that prevent axonal regrowth. Myelin growth inhibits and their common receptor have been identified as targets in the treatment of damage to the CNS.We have recently determined the NMR structure of one of the myelin growth inhibitors, the neurite outgrowth inhibitor (Nogo). We studied the structure of this protein alone and in the presence of dodecylphosphocholine micelles to mimic the natural cell Membrane environment. Using several paramagnetic probes, we have defined portions of the growth inhibitor that are accessible to solvent (and consequently the Nogo receptor). Mutagenesis probed through phage-display confirms that the positions predicted to be extra-cellular are sensitive to receptor binding. Using computational docking methods and the mutagenesis results, we calculated the optimal protein-protein Interface between our structure of Nogo and the Nogo receptor. The structure of Nogo and the predicted Nogo/receptor inhibitory complex structure will be presented.Funding was provided by: NIH 1R01GM078528-01 andCal Roman Reed Res Fund RR03-072, +114, +155