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Israel Silman - One of the best experts on this subject based on the ideXlab platform.

  • Torpedo Californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4d motif
    Protein Science, 2021
    Co-Authors: Israel Silman, Joel L. Sussman, Valery L Shnyrov, Esther Roth, Yacov Ashani, Anne Nicolas, Lev Weiner
    Abstract:

    Stabilization of Torpedo Californica acetylcholinesterase by the divalent cations Ca+2 , Mg+2 , and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several water molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of RMSD values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Protein_Science:3.

  • Torpedo Californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4d motif
    Protein Science, 2021
    Co-Authors: Israel Silman, Joel L. Sussman, Valery L Shnyrov, Esther Roth, Yacov Ashani, Anne Nicolas, Lev Weiner
    Abstract:

    Stabilization of Torpedo Californica acetylcholinesterase by the divalent cations Ca+2 , Mg+2 and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several waters molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of rmsd values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. This article is protected by copyright. All rights reserved.

  • Polyproline-rich peptides associated with Torpedo Californica acetylcholinesterase tetramers.
    Chemico-biological interactions, 2020
    Co-Authors: Lilly Toker, Lawrence M Schopfer, Israel Silman, Tzviya Zeev-ben-mordehai, Joel L. Sussman, Oksana Lockridge
    Abstract:

    Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo Californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo Californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.

  • Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo Californica
    2014
    Co-Authors: Gertraud Koellner, Israel Silman, Joel L. Sussman, Gitay Kryger, Charles B Millard, Thomas Steiner
    Abstract:

    *Corresponding authors Buried water molecules and the water molecules in the active-site gorge are analyzed for ®ve crystal structures of acetylcholinesterase from Torpedo Californica in the resolution range 2.2-2.5 AÊ (native enzyme, and four inhibitor complexes). A total of 45 buried hydration sites are identi®ed, which are populated with between 36 and 41 water molecules. About half of the buried water is located in a distinct region neighboring the active-site gorge. Most of the buried water molecules are very well conserved among the ®ve structures, and have low displacement parameters, B, of magnitudes similar to those of the main-chain atoms of the central b-sheet structure. The active-site gorge of the native enzyme is ®lled with over 20 water molecules, which have poor hydrogen-bond coordination with an average of 2.9 polar contacts per water molecule. Upon ligand binding, distinct groups of these water molecules are displaced, whereas the others remain in positions similar to those that they occupy in th

  • structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo Californica acetylcholinesterase
    Protein Science, 2012
    Co-Authors: Esther Roth, Israel Silman, Joel L. Sussman, Valery L Shnyrov, Yacov Ashani, Yechun Xu, Lev Weiner
    Abstract:

    The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo Californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic “anionic” subsite (CAS), the peripheral “anionic” subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both “anionic” subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ∼8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

Joel L. Sussman - One of the best experts on this subject based on the ideXlab platform.

  • Torpedo Californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4d motif
    Protein Science, 2021
    Co-Authors: Israel Silman, Joel L. Sussman, Valery L Shnyrov, Esther Roth, Yacov Ashani, Anne Nicolas, Lev Weiner
    Abstract:

    Stabilization of Torpedo Californica acetylcholinesterase by the divalent cations Ca+2 , Mg+2 and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several waters molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of rmsd values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. This article is protected by copyright. All rights reserved.

  • Torpedo Californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4d motif
    Protein Science, 2021
    Co-Authors: Israel Silman, Joel L. Sussman, Valery L Shnyrov, Esther Roth, Yacov Ashani, Anne Nicolas, Lev Weiner
    Abstract:

    Stabilization of Torpedo Californica acetylcholinesterase by the divalent cations Ca+2 , Mg+2 , and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several water molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of RMSD values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Protein_Science:3.

  • Polyproline-rich peptides associated with Torpedo Californica acetylcholinesterase tetramers.
    Chemico-biological interactions, 2020
    Co-Authors: Lilly Toker, Lawrence M Schopfer, Israel Silman, Tzviya Zeev-ben-mordehai, Joel L. Sussman, Oksana Lockridge
    Abstract:

    Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo Californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo Californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.

  • Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo Californica
    2014
    Co-Authors: Gertraud Koellner, Israel Silman, Joel L. Sussman, Gitay Kryger, Charles B Millard, Thomas Steiner
    Abstract:

    *Corresponding authors Buried water molecules and the water molecules in the active-site gorge are analyzed for ®ve crystal structures of acetylcholinesterase from Torpedo Californica in the resolution range 2.2-2.5 AÊ (native enzyme, and four inhibitor complexes). A total of 45 buried hydration sites are identi®ed, which are populated with between 36 and 41 water molecules. About half of the buried water is located in a distinct region neighboring the active-site gorge. Most of the buried water molecules are very well conserved among the ®ve structures, and have low displacement parameters, B, of magnitudes similar to those of the main-chain atoms of the central b-sheet structure. The active-site gorge of the native enzyme is ®lled with over 20 water molecules, which have poor hydrogen-bond coordination with an average of 2.9 polar contacts per water molecule. Upon ligand binding, distinct groups of these water molecules are displaced, whereas the others remain in positions similar to those that they occupy in th

  • structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo Californica acetylcholinesterase
    Protein Science, 2012
    Co-Authors: Esther Roth, Israel Silman, Joel L. Sussman, Valery L Shnyrov, Yacov Ashani, Yechun Xu, Lev Weiner
    Abstract:

    The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo Californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic “anionic” subsite (CAS), the peripheral “anionic” subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both “anionic” subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ∼8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

Lev Weiner - One of the best experts on this subject based on the ideXlab platform.

  • Torpedo Californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4d motif
    Protein Science, 2021
    Co-Authors: Israel Silman, Joel L. Sussman, Valery L Shnyrov, Esther Roth, Yacov Ashani, Anne Nicolas, Lev Weiner
    Abstract:

    Stabilization of Torpedo Californica acetylcholinesterase by the divalent cations Ca+2 , Mg+2 and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several waters molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of rmsd values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. This article is protected by copyright. All rights reserved.

  • Torpedo Californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4d motif
    Protein Science, 2021
    Co-Authors: Israel Silman, Joel L. Sussman, Valery L Shnyrov, Esther Roth, Yacov Ashani, Anne Nicolas, Lev Weiner
    Abstract:

    Stabilization of Torpedo Californica acetylcholinesterase by the divalent cations Ca+2 , Mg+2 , and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several water molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of RMSD values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Protein_Science:3.

  • structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo Californica acetylcholinesterase
    Protein Science, 2012
    Co-Authors: Esther Roth, Israel Silman, Joel L. Sussman, Valery L Shnyrov, Yacov Ashani, Yechun Xu, Lev Weiner
    Abstract:

    The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo Californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic “anionic” subsite (CAS), the peripheral “anionic” subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both “anionic” subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ∼8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

  • targeted oxidation of Torpedo Californica acetylcholinesterase by singlet oxygen
    Photochemistry and Photobiology, 2011
    Co-Authors: Lev Weiner, Esther Roth, Israel Silman
    Abstract:

    The photosensitizer, methylene blue (MB), is a strong reversible inhibitor of Torpedo Californica acetylcholinesterase (AChE) in the dark. Under illumination it causes irreversible inactivation. Loss of fluorescence of the singlet oxygen ((1)O(2)) trap, 9,10-dimethylanthracene, was retarded in the presence of AChE, and the rate of photo-inactivation was increased in the presence of D(2)O, indicating that inactivation was due to (1)O(2) generated by the photosensitizer. CD revealed slightly reduced far-UV ellipticity, and slightly enhanced binding of an amphiphilic probe, indicating limited unfolding of the photo-oxidized AChE. However, both near-UV ellipticity and intrinsic fluorescence were markedly reduced, suggesting photo-oxidative damage to tryptophans, (Trp) supported by appearance of novel emission peaks ascribed to N'-formylkynurenine and/or kynurenine. Like other partially unfolded forms, the photo-oxidized AChE was sensitive to proteolysis. Photosensitized inactivation produced exclusively chemically cross-linked dimers, whereas irradiation of a partially unfolded state generated higher-order oligomers. The active-site gorge of AChE contains Trp in inhibitor-binding sites that might be targets for photo-oxidation. Indeed, reversible inhibitors retard photo-inactivation, and photo-inactivation destroys their binding sites. An excess of AChE protects paraoxonase from photo-inactivation by sequestering the photosensitizer. Affinity photo-oxidation of AChE by MB thus provides a valuable model for studying site-specific photo-inactivation of enzymes in both fundamental and clinical contexts.

  • stabilization of Torpedo Californica acetylcholinesterase by reversible inhibitors
    Biochemistry, 2009
    Co-Authors: Lev Weiner, Valery L Shnyrov, Leonid Konstantinovskii, Esther Roth, Yacov Ashani, Israel Silman
    Abstract:

    The dimeric form of Torpedo Californica acetylcholinesterase provides a valuable experimental system for studying transitions between native, partially unfolded, and unfolded states since long-lived partially unfolded states can be generated by chemical modification of a nonconserved buried cysteine residue, Cys 231, by denaturing agents, by oxidative stress, and by thermal inactivation. Elucidation of the 3D structures of complexes of Torpedo Californica acetylcholinesterase with a repertoire of reversible inhibitors permits their classification into three categories: (a) active-site directed inhibitors, which interact with the catalytic anionic subsite, at the bottom of the active-site gorge, such as edrophonium and tacrine; (b) peripheral anionic site inhibitors, which interact with a site at the entrance to the gorge, such as propidium and d-tubocurarine; and (c) elongated gorge-spanning inhibitors, which bridge the two sites, such as BW284c51 and decamethonium. The effects of these three categories o...

Jose A Lasaldedominicci - One of the best experts on this subject based on the ideXlab platform.

  • sequential purification and characterization of Torpedo Californica nachr dc supplemented with chs for high resolution crystallization studies
    Analytical Biochemistry, 2020
    Co-Authors: Rafael Maldonadohernandez, Orestes Quesada, Jose A Lasaldedominicci, Jose O Colonsaez
    Abstract:

    Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo Californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification. We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of β-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-β-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-β-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.

  • lipid profile and functionality of nicotinic aceytilcholine receptor from Torpedo Californica solubillized with cyclofos detergent family
    Biophysical Journal, 2017
    Co-Authors: Orestes Quesada, Jose O Colonsaez, Carol Gonzalez, Rafael Maldonado, Irvin Rosado, Alan Espinal, Jesus Acevedo, Jose A Lasaldedominicci
    Abstract:

    The first step for reconstitution in attempts to membrane protein crystallization is the solubilization process by detergents. During this process, preservation of the native protein structure and adjacent lipid environments is crucial for activity and stability. The detergent disruption of lipid-lipid, lipid-protein interactions alter the aggregation state, stability, and functionality of the native structure. The effect is more severe when protein activity is lipid dependent. The molecular composition of all detected phospholipid classes from affinity-purified acetylcholine receptors detergent complexes (nAChR-DCs) from Torpedo Californica (Tc) solubilized using a homologous series of Cyclo Foscholine (CF) detergents were analyzed by Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). We took advantage of Xenopus laevis oocytes expression system to access the nAChR-DCs activity by means of two electrode voltage clamp (TEVC) technique. UPLC/ESI/MS/MS results shown a phospholipid composition favored by phosphatidylcholine and sphingomyelin species for the three nAChR-DC studied. However, the molecular species in each nAChr-DC and cholesterol content were in some degree, quite different despite that the only detergent structural difference is 1 or 2 methylene groups. Injection of oocytes with Tc nAChRs-DCs solubilized with CF family, differing in the length of their acyl chain, resulted in responses with amplitudes that inversely depend on acyl chain length. CF-4 displayed the highest mean amplitudes in this family (−200 ± 60 nA) which is about 80% of the current produced when normalized to the mean current of the crude membrane. The longer side chain, CF-6 and CF-7 produced an insignificant current compared to the crude membrane. These findings will provide a lipidomic strategy to prepare nAChR-DCs suitable for structural studies.

  • effects of lipid analog detergent solubilization on the functionality and lipidic cubic phase mobility of the Torpedo Californica nicotinic acetylcholine receptor
    The Journal of Membrane Biology, 2011
    Co-Authors: Luis F Padillamorales, Claudio L Moralesperez, Pamela C De La Cruzrivera, Guillermo Asmarrovira, Orestes Quesada, Carlos A Baezpagan, Jose A Lasaldedominicci
    Abstract:

    Over the past three decades, the Torpedo Californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β2-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.

  • characterization of nicotinic acetylcholine receptor from Torpedo Californica uding lipid analog detergents
    Biophysical Journal, 2011
    Co-Authors: Luis F Padillamorales, Claudio L Moralesperez, Pamela C De La Cruzrivera, Luis M Lopezcruz, Guillermo Asmarrovira, Raymond C Stevens, Orestes Quesada, Jose A Lasaldedominicci
    Abstract:

    In the present study, we characterized the effect of detergent solubilization and affinity column purification on Torpedo Californica nicotinic acetylcholine receptor (nAChR) by using a series of lipid-like detergent with similar acyl composition to the most abundant fatty acid found in the native tissue of Torpedo (18:1, 18:0, 16:1, 16:0) as well as cholesterol-analog detergents. Fatty acid analogs included members of the Fos-choline (FC) family of detergents (FC-12, −14, −16 and lyso-FC-16), while cholesterol analogs were represented by cholate, taurocholate and CHAPS. Each detergent was used to solubilize and purify the nAChR using established affinity column protocols, followed by analytical size exclusion chromatography (A-SEC) to probe the stability and aggregation state of the nAChR in solution, as well as planar lipid bilayers to probe ion channel function. The overall results showed that the stability and function of the solubilized and purified receptor is preserved in lipid-analog detergents that have acyl chains similar to the most abundant lipid (18:1, 18:0, 16:1, and 16:0) in the endogenous Torpedo lipid environment, providing a suitable set of detergents for future structural studies. We also performed lipidic cubic phase (LCP) fluorescence recovery after photo bleaching (FRAP) experiments using fluorescently-tagged-Torpedo nAChR solubilized and affinity purified in FC-12, −14 and lyso-FC-16 detergents to estimate the mobile fraction and diffusion coefficient with the goal of using these data to perform LCP crystallization trials within a narrower crystallization space. These detergents display a linear increase in mobile fraction and diffusion coefficient (LFC-16>FC-12>FC-14) which is inversely proportional to the percentage of dimmer in these detergents (FC-14>FC-12>LFC-16). These experiments will serve to establish correlations to define suitable conditions for nAChR crystallization in LCP.

  • efficient isolation and characterization of nicotinic acetylcholine receptor from Torpedo Californica using lipid analog detergents
    Biophysical Journal, 2010
    Co-Authors: Luis F Padillamorales, Claudio L Moralesperez, Pamela C De La Cruzrivera, Luis M Lopezcruz, Guillermo Asmarrovira, Raymond C Stevens, Orestes Quesada, Jose A Lasaldedominicci
    Abstract:

    The effect of detergent solubilization on nicotinic acetylcholine receptor (nAChR) function has been extensively studied by several laboratories with the ultimate goal of characterizing the dynamic detergent-lipid-protein interactions of functional nAChR in both native and reconstituted membranes, as well as the detergent-solubilized state. These studies have provided substantial data on suitable detergents for solubilization, purification and functional reconstitution of the nAChR obtained from the electric organ of Torpedo Californica electric rays. However, the molecular mechanisms by which particular detergents influence nAChR function remain poorly understood. In the present study, we characterized the effect of detergent solubilization and affinity column purification on Torpedo nAChR by using a series of lipid-like detergent with similar acyl composition to the most abundant fatty acid found in the native tissue of Torpedo (16:0, 18:0, 16:1 16:0)as well as cholesterol- analog detergents. . Fatty acid analogs included members of the Fos-choline (FC) family of detergents (FC-12, −14, −16 and lyso-FC), while cholesterol analogs were represented by cholate, taurocholate and CHAPS. Each detergent was used to s solubilize and purify the nAChR using established affinity column protocols, followed by analytical size exclusion chromatography (A-SEC) to probe the stability and aggregation state of the nAChR in solution, as well as planar lipid bilayers to probe ion channel function. The overall results showed that the stability and function of the solubilized and purified receptor is preserved in lipid-analog detergents that have acyl chains similar to the most abundant lipid (16:0, 18:0, 16:1 16:0) in the endogenous Torpedo lipid environment, providing a suitable set of detergents for future studies. Supported by NIH grants, RISE, FIPI, 2RO1GM56371-12 and 2U54NS43011.

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  • kinetic and structural studies on the interactions of Torpedo Californica acetylcholinesterase with two donepezil like rigid analogues
    Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
    Co-Authors: Rosanna Caliandro, Alessandro Pesaresi, Luca Cariati, Antonio Procopio, Manuela Oliverio, Doriano Lamba
    Abstract:

    AbstractAcetylcholinesterase inhibitors were introduced for the symptomatic treatment of Alzheimer’s disease (AD). Among the currently approved inhibitors, donepezil (DNP) is one of the most preferred choices in AD therapy. The X-ray crystal structures of Torpedo Californica AChE in complex with two novel rigid DNP-like analogs, compounds 1 and 2, have been determined. Kinetic studies indicated that compounds 1 and 2 show a mixed-type inhibition against TcAChE, with Ki values of 11.12 ± 2.88 and 29.86 ± 1.12 nM, respectively. The DNP rigidification results in a likely entropy-enthalpy compensation with solvation effects contributing primarily to AChE binding affinity. Molecular docking evidenced the molecular basis for the binding of compounds 1 and 2 to the active site of β-secretase-1. Overall, these simplified DNP derivatives may represent new structural templates for the design of lead compounds for a more effective therapeutic strategy against AD by foreseeing a dual AChE and BACE-1 inhibitory activity.

  • kinetics of Torpedo Californica acetylcholinesterase inhibition by bisnorcymserine and crystal structure of the complex with its leaving group
    Biochemical Journal, 2012
    Co-Authors: C Bartolucci, Jure Stojan, Qiansheng Yu, Nigel H Greig, Doriano Lamba
    Abstract:

    Natural and synthetic carbamates act as pseudo-irreversible inhibitors of AChE (acetylcholinesterase) as well as BChE (butyrylcholinesterase), two enzymes involved in neuronal function as well as in the development and progression of AD (Alzheimer9s disease). The AChE mode of action is characterized by a rapid carbamoylation of the active-site Ser 200 with release of a leaving group followed by a slow regeneration of enzyme action due to subsequent decarbamoylation. The experimental AD therapeutic bisnorcymserine, a synthetic carbamate, shows an interesting activity and selectivity for BChE, and its clinical development is currently being pursued. We undertook detailed kinetic studies on the activity of the carbamate bisnorcymserine with Tc ( Torpedo Californica ) AChE and, on the basis of the results, crystallized the complex between Tc AChE and bisnorcymserine. The X-ray crystal structure showed only the leaving group, bisnoreseroline, trapped at the bottom of the aromatic enzyme gorge. Specifically, bisnoreseroline interacts in a non-covalent way with Ser 200 and His 440 , disrupting the existing interactions within the catalytic triad, and it stacks with Trp 84 at the bottom of the gorge, giving rise to an unprecedented hydrogen-bonding contact. These interactions point to a dominant reversible inhibition mechanism attributable to the leaving group, bisnoreseroline, as revealed by kinetic analysis.

  • probing Torpedo Californica acetylcholinesterase catalytic gorge with two novel bis functional galanthamine derivatives
    Journal of Medicinal Chemistry, 2010
    Co-Authors: Cecilia Bartolucci, Ulrich Jordis, Gregor Fels, Lars A Haller, Doriano Lamba
    Abstract:

    N-Piperidinopropyl-galanthamine (2) and N-saccharinohexyl-galanthamine (3) were used to investigate interaction sites along the active site gorge of Torpedo Californica actylcholinesterase (TcAChE). The crystal structure of TcAChE-2 solved at 2.3 A showed that the N-piperidinopropyl group in 2 is not stretched along the gorge but is folded over the galanthamine moiety. This result was unexpected because the three carbon alkyl chain is just long enough for the bulky piperidine group to be placed above the bottleneck (Tyr121, Phe330) midway down the gorge. The crystal structure of TcAChE-3 at 2.2 A confirmed that a dual interaction with the sites at the bottom, and at the entrance of the gorge, enhances inhibitory activity: a chain of six carbon atoms has, in this class of derivatives, the correct length for optimal interactions with the peripheral anionic site (PAS).

  • structural determinants of Torpedo Californica acetylcholinesterase inhibition by the novel and orally active carbamate based anti alzheimer drug ganstigmine chf 2819
    Journal of Medicinal Chemistry, 2006
    Co-Authors: Cecilia Bartolucci, Mariacristina Siotto, Eleonora Ghidini, Gabriele Amari, Pier Tonino Bolzoni, Marco Racchi, Gino Villetti, Maurizio Delcanale, Doriano Lamba
    Abstract:

    Ganstigmine is an orally active, geneserine derived, carbamate-based acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease. The crystal structure of the ganstigmine conjugate with Torpedo Californica acetylcholinesterase (TcAChE) has been determined at 2.40 A resolution, and a detailed structure-based analysis of the in vitro and ex vivo anti-AChE activity by ganstigmine and by new geneserine derivatives is presented. The carbamoyl moiety is covalently bound to the active-site serine, whereas the leaving group geneseroline is not retained in the catalytic pocket. The nitrogen atom of the carbamoyl moiety of ganstigmine is engaged in a key hydrogen-bonding interaction with the active site histidine (His440). This result offers an explanation for the inactivation of the catalytic triad and may account for the long duration of action of ganstigmine in vivo. The 3D structure also provides a structural framework for the design of compounds with improved binding affinity and pharmacological properties.

  • accurate prediction of the bound conformation of galanthamine in the active site of Torpedo Californica acetylcholinesterase using molecular docking
    Journal of Molecular Graphics & Modelling, 2001
    Co-Authors: Christian Pilger, Cecilia Bartolucci, Doriano Lamba, Alexander Tropsha, Gregor Fels
    Abstract:

    Abstract The alkaloid (−)-galanthamine is known to produce significant improvement of cognitive performances in patients with the Alzheimer’s disease. Its mechanism of action involves competitive and reversible inhibition of acetylcholinesterase (AChE). Herein, we correctly predict the orientation and conformation of the galanthamine molecule in the active site of AChE from Torpedo Californica (TcAChE) using a combination of rigid docking and flexible geometry optimization with a molecular mechanics force field. The quality of the predicted model is remarkable, as indicated by the value of the RMS deviation of ∼0.5A when compared with the crystal structure of the TcAChE-galanthamine complex. A molecular model of the complex between TcAChE and a galanthamine derivative, SPH1107, with a long chain substituent on the nitrogen has been generated as well. The side chain of this ligand is predicted to extend along the enzyme active site gorge from the anionic subsite, at the bottom, to the peripheral anionic site, at the top. The docking procedure described in this paper can be applied to produce models of ligand-receptor complexes for AChE and other macromolecular targets of drug design.