The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Takeshi Murata - One of the best experts on this subject based on the ideXlab platform.
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structure and function of vacuolar na translocating atpase in enterococcus hirae
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Yoshimi Kakinuma, Ichiro Yamato, Takeshi MurataAbstract:A Na+-translocating ATPase was discovered in a gram-positive bacterium Enterococcus hirae. Our biochemical and molecular biological studies revealed that this Na+-ATPase belongs to the vacuolar-type enzyme. Purified Na+-ATPase consisted of nine subunits: NtpA, B, C, D, E, F, G, I, and K; reconstituted proteoliposomes showed ATP-driven electrogenic Na+ translocation. All these subunits were encoded by the ntp operon: ntpFIKECGABDHJ. The deduced amino acid sequences of the major subunits, A, B, and K (16 kDa Proteolipid), were highly similar to those of A, B, and Proteolipid subunits of vacuolar ATPases, although the similarities of other subunits were moderate. The ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at transcriptional level by intracellular Na+ as the signal. Two related cation pumps, vacuolar Na+-ATPase and F0F1, H+-ATPase, coexist in this bacterium.
Gordon Tollin - One of the best experts on this subject based on the ideXlab platform.
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different structural states of the Proteolipid membrane are produced by ligand binding to the human δ opioid receptor as shown by plasmon waveguide resonance spectroscopy
Molecular Pharmacology, 2004Co-Authors: Isabel D Alves, Zdzislaw Salamon, Gordon Tollin, Scott Cowell, Savitha Devanathan, Victor J HrubyAbstract:Understanding structure-function relationships and mechanisms of signal transduction in G-protein-coupled receptors (GPCRs) is becoming increasingly important, both as a fundamental problem in membrane biology and as a consequence of their central role as pharmacological targets. Their integral membrane nature and rather low natural abundance present many challenging problems. Using a recently developed technique, plasmon-waveguide resonance (PWR) spectroscopy, we investigated the structural changes accompanying the binding of ligands to the human δ-opioid receptor (hDOR) immobilized in a solid-supported lipid bilayer. This highly sensitive technique can directly monitor changes in mass density, conformation, and orientation occurring in such thin Proteolipid films. Without requiring labeling protocols, PWR allows the direct determination of binding constants in a system very close to the receptor9s natural environment. In the present study, conformational changes of a Proteolipid membrane containing the hDOR were investigated upon binding of a variety of peptide and nonpeptide agonists, partial agonists, antagonists, and inverse agonists. Distinctly different structural states of the membrane were observed upon binding of each of these classes of ligands, reflecting different receptor conformational states, and the formation of each state was characterized by different kinetic properties. Binding constants, obtained by quantifying the extent of conformational change as a function of the amount of ligand bound, were in good agreement with published values determined by radiolabeling methods. The results provide new insights into ligand-induced GPCR functioning and illustrate a powerful new protocol for drug development.
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optical anisotropy in lipid bilayer membranes coupled plasmon waveguide resonance measurements of molecular orientation polarizability and shape
Biophysical Journal, 2001Co-Authors: Zdzislaw Salamon, Gordon TollinAbstract:Abstract The birefringence and linear dichroism of anisotropic thin films such as Proteolipid membranes are related to molecular properties such as polarizability, shape, and orientation. Coupled plasmon-waveguide resonance (CPWR) spectroscopy is shown in the present work to provide a convenient means of evaluating these parameters in a single lipid bilayer. This is illustrated by using 1–10mol % of an acyl chain chromophore-labeled phosphatidylcholine (PC) incorporated into a solid-supported PC bilayer deposited onto a hydrated silica surface. CPWR measurements were made of refractive index and extinction coefficient anisotropies with two exciting light wavelengths, one of which is absorbed by the chromophore and one of which is not. These results were used to calculate longitudinal and transverse molecular polarizabilities, the orientational order parameter and average angle between the longitudinal axis of the lipid molecule and the membrane normal, and the molecular shape factors of the lipid molecules. The values thereby obtained are in excellent agreement with parameters determined by other techniques, and provide a powerful tool for analyzing lipid-protein, protein-protein, and protein-ligand interactions in Proteolipid films.
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coupled plasmon waveguide resonance spectroscopy studies of the cytochrome b6f plastocyanin system in supported lipid bilayer membranes
Biophysical Journal, 1998Co-Authors: Zdzislaw Salamon, D Huang, William A Cramer, Gordon TollinAbstract:Abstract The incorporation of cytochrome (cyt) b 6 f into a solid-supported planar egg phosphatidylcholine (PC) bilayer membrane and complex formation with plastocyanin have been studied by a variant of surface plasmon resonance called coupled plasmon-waveguide resonance (CPWR) spectroscopy, developed in our laboratory. CPWR combines greatly enhanced sensitivity and spectral resolution with direct measurement of anisotropies in refractive index and optical extinction coefficient, and can therefore probe structural properties of lipid-protein and protein-protein interactions. Cyt b 6 f incorporation into the membrane proceeds in two stages. The first occurs at low protein concentration and is characterized by an increase in total Proteolipid mass without significant changes in the molecular order of the system, as demonstrated by shifts of the resonance position to larger incident angles without changing the refractive index anisotropy. The second stage, occurring at higher protein concentrations, results in a decrease in both the mass density and the molecular order of the system, evidenced by shifts of the resonance position to smaller incident angles and a large decrease in the membrane refractive index anisotropy. Plastocyanin can bind to such a Proteolipid system in three different ways. First, the addition of plastocyanin before the second stage of b 6 f incorporation begins results in complex formation between the two proteins with a K D of ∼10 μ M and induces structural changes in the membrane that are similar to those occurring during the second stage of complex incorporation. The addition of larger amounts of plastocyanin under these conditions leads to nonspecific binding to the lipid phase with a K D of ∼180 μ M. Finally, the addition of plastocyanin after the completion of the second phase of b 6 f incorporation results in tighter binding between the two proteins ( K D ≈ 1 μ M). Quantitation of the binding stoichiometry indicates that two plastocyanin molecules bind tightly to the dimeric form of the cyt b 6 f complex, assuming random insertion of the cytochrome into the bilayer. The structural basis for these results and formation of the Proteolipid membrane are discussed.
Wah Chiu - One of the best experts on this subject based on the ideXlab platform.
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the 3 5 a cryoem structure of nanodisc reconstituted yeast vacuolar atpase vo proton channel
Molecular Cell, 2018Co-Authors: Soung Hun Roh, Nicholas J Stam, Corey F Hryc, Sergio Couohcardel, Grigore Pintilie, Wah Chiu, Stephan WilkensAbstract:The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-A resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+-ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the Proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport. Moreover, we discovered that the C terminus of the assembly factor Voa1 is an integral component of mature Vo. Voa1's C-terminal transmembrane α helix is bound inside the Proteolipid ring, where it contributes to the stability of the complex. Our structure rationalizes possible mechanisms by which mutations in human Vo can result in disease phenotypes and may thus provide new avenues for therapeutic interventions.
Soung Hun Roh - One of the best experts on this subject based on the ideXlab platform.
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the 3 5 a cryoem structure of nanodisc reconstituted yeast vacuolar atpase vo proton channel
Molecular Cell, 2018Co-Authors: Soung Hun Roh, Nicholas J Stam, Corey F Hryc, Sergio Couohcardel, Grigore Pintilie, Wah Chiu, Stephan WilkensAbstract:The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-A resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+-ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the Proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport. Moreover, we discovered that the C terminus of the assembly factor Voa1 is an integral component of mature Vo. Voa1's C-terminal transmembrane α helix is bound inside the Proteolipid ring, where it contributes to the stability of the complex. Our structure rationalizes possible mechanisms by which mutations in human Vo can result in disease phenotypes and may thus provide new avenues for therapeutic interventions.
Yoshimi Kakinuma - One of the best experts on this subject based on the ideXlab platform.
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indispensable glutamic acid residue 139 of ntpk Proteolipid in the reaction of vacuolar na translocating atpase in enterococcus hirae
Bioscience Biotechnology and Biochemistry, 1999Co-Authors: Kazuma Takase, Ichiro Yamato, Kazuei Igarashi, Yoshimi KakinumaAbstract:Enterococcus hirae vacuolar ATPase catalyzes translocation of Na+ or Li+ coupled with ATP hydrolysis. It is suggested that the glutamic acid residue (Glu139) of NtpK Proteolipid subunit of this multisubunit enzyme is the binding site of these ions for translocation. Here we established a complementation system for the ntpK gene with its deletion mutant, and found that the ATPase activity disappeared upon replacement of Glu139 by aspartic acid. The side-chain length of this acidic residue of NtpK is thus important for this ATPase reaction.
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structure and function of vacuolar na translocating atpase in enterococcus hirae
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Yoshimi Kakinuma, Ichiro Yamato, Takeshi MurataAbstract:A Na+-translocating ATPase was discovered in a gram-positive bacterium Enterococcus hirae. Our biochemical and molecular biological studies revealed that this Na+-ATPase belongs to the vacuolar-type enzyme. Purified Na+-ATPase consisted of nine subunits: NtpA, B, C, D, E, F, G, I, and K; reconstituted proteoliposomes showed ATP-driven electrogenic Na+ translocation. All these subunits were encoded by the ntp operon: ntpFIKECGABDHJ. The deduced amino acid sequences of the major subunits, A, B, and K (16 kDa Proteolipid), were highly similar to those of A, B, and Proteolipid subunits of vacuolar ATPases, although the similarities of other subunits were moderate. The ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at transcriptional level by intracellular Na+ as the signal. Two related cation pumps, vacuolar Na+-ATPase and F0F1, H+-ATPase, coexist in this bacterium.
