Binding of Iodide to Arthromyces ramosus Peroxidase Investigated with X-ray Crystallographic Analysis, 1H and 127I NMR Spectroscopy, and Steady-state Kinetics

Fukuyama, Keiichi; Sato, Koichi; Itakura, Hikaru; Takahashi, Shunsuke; Hosoya, Toshihiko

doi:10.1074/jbc.272.9.5752

Cited by 32 publications

(23 citation statements)

References 52 publications

(49 reference statements)

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“…Comparison of the Rates of Backsliding and Translocation —Previous measurements of translocation rates were performed by adding translocation substrate to inverted vesicles or reconstituted proteoliposomes (4, 6, 7, 24). However, these measurements include the time required for the binding of substrate to SecA and SecY and for other steps and therefore do not report the true rate of polypeptide movement through the channel.…”

Section: Resultsmentioning

confidence: 99%

Analysis of Polypeptide Movement in the SecY Channel during SecA-mediated Protein Translocation

Erlandson

Or²,

Osborne³

et al. 2008

Journal of Biological Chemistry

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In bacteria most secretory proteins are transported across the plasma membrane by the interplay of the ATPase SecA with the translocation channel formed by the SecY complex; SecA uses cycles of ATP hydrolysis to “push” consecutive segments of a polypeptide substrate through the channel. Here we have addressed the mechanism of this process by following the fate of stalled translocation intermediates. These were generated by using a polypeptide substrate containing a bulky disulfide-bonded loop, thus preventing the final residues from passing through the channel. Protease protection experiments showed that the intermediates were stable in the presence of ATP and could complete translocation once the block was removed. The translocation intermediate was also stable when SecA associated with ATPγS, a poorly hydrolyzable ATP analog, or ADP plus AlF4, which mimics the transition state during ATP hydrolysis. In contrast, when SecA was in its ADP-bound state, the translocating polypeptide moved back into the cytosol, as indicated by the disappearance of the protected fragment. Backsliding was not significantly altered by deletion of the plug domain, a short helix in the center of the SecY channel, but it was slowed down when changes were introduced into the pore ring, the constriction of the hourglass-shaped channel. In all cases, backsliding was significantly slower than forward translocation. Together, these data suggest that SecA binds the polypeptide chain in its ATP state and releases it in the ADP state. The channel itself does not bind the polypeptide chain but provides “friction” that minimizes backsliding when ADP-bound SecA resets to “grab” the next segment of the substrate.

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Section: Resultsmentioning

confidence: 99%

Analysis of Polypeptide Movement in the SecY Channel during SecA-mediated Protein Translocation

Erlandson

Or²,

Osborne³

et al. 2008

Journal of Biological Chemistry

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“…For further details, see "Discussion." (14,20). Thus, the close proximity with the negative charge of the propionate could give this H 2 O molecule(s) a strong ionic character with a very high pK a value.…”

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confidence: 98%

“…The crystal structures of both peroxidases have been solved (14,20), and the protein from C. cinereus has been thoroughly characterized by spectroscopic techniques (21)(22)(23). This study has focused on the role of Asp 245 H-bonded to the proximal His 183 in the redox and ligand binding properties as a function of pH.…”

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confidence: 99%

Relationships of Ligand Binding, Redox Properties, and Protonation in Coprinus cinereus Peroxidase

Ciaccio

Rosati

Sanctis

et al. 2003

Journal of Biological Chemistry

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The pH dependence of the redox potentials and kinetics for CO association and dissociation was determined between pH 3.0 and 13.0 at 25°C for the wild-type Coprinus cinereus fungal peroxidase and for a site-directed mutant in which Asp 245, which is H-bonded to N ␦ of the imidazole of the proximal His 183, was substituted with Asn. The determination of these functional properties allowed this information to be merged in a self-consistent fashion and to formulate for the first time a complete scheme employing the minimum number of groups required to describe the whole proton-linked behavior of both redox and ligand binding properties. The overall pH dependence can be accounted for by four redox-and ligand-linked groups. The proximal H-bond, which is strictly conserved in all peroxidases, will still be present in the site-specific mutant, but will no longer have an ionic character, and this event will bring about an alteration of redox equilibria and CO binding kinetics, envisaging a relevant role played by this H-bond also in modulating redox properties and ligand binding equilibria.

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“…This showed that the binding of iodide depends on protonation of an amino acid residue with a pKa of around 5.3, which was presumed to be the distal histidine (His56), which is 7.8 Å away from the iodide ion [318].…”

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confidence: 99%

NMR-Active Nuclei for Biological and Biomedical Applications

Patching¹

2016

J Diagn Imaging Ther

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Nuclear magnetic resonance (NMR) spectroscopy is a principal well-established technique for analysis of chemical, biological, food and environmental samples. This article provides an overview of the properties and applications of NMR-active nuclei (39 nuclei of 33 different elements) used in NMR measurements (solution-and solid-state NMR, magnetic resonance spectroscopy, magnetic resonance imaging) with biological and biomedical systems and samples. The samples include biofluids, cells, tissues, organs or whole body from different organisms (humans, animals, bacteria, fungi, plants) for detecting and quantifying metabolites or environmental samples (water, soils, sediments). Isolated biomolecules (peptides, proteins, nucleic acids) can be analysed for elucidation of atomic-resolution structure, conformation and dynamics and for characterisation of ligand and drug binding, and of protein-ligand, protein-protein and protein-nucleic acid interactions. NMR can be used for drug screening and pharmacokinetics and to provide information in the design and discovery of new drugs. NMR can also measure translocation of ions and small molecules across lipid bilayers and membranes, characterise structure, phase behaviour and dynamics of membranes and elucidate atomic-resolution structure, orientation and dynamics of membrane-embedded peptides and proteins.Keywords: biological and biomedical applications; drug screening; dynamics; magnetic resonance imaging; membrane proteins; metabolomics; MRI; NMR-active nuclei; nuclear magnetic resonance; protein structure INTRODUCTION1 UCLEAR magnetic resonance (NMR) spectroscopy is one of the principal techniques used for analysis of biological and biomedical systems and samples. This can include the identification, quantification and monitoring of ions, small molecules and biomolecules in studies of metabolism and biological function in human and animal cells and tissues, bacterial cells and spores, fungi and plants. Similar types of measurements can be performed on environmental samples such as water, soils and sediment. NMR is able to elucidate atomic-resolution structure, conformation, molecular mechanism, dynamics and exchange processes (on timescales of picoseconds to seconds) in biomolecules, especially peptides, proteins and nucleic acids. NMR can be used for the observation, quantification and characterisation of ligand and drug binding to biomolecules, and for characterisation of ligandprotein, protein-protein and protein-nucleic acid interactions. NMR can be used for drug screening and it can acquire structural, binding and kinetic information for the design and discovery of new drugs. It can then monitor the absorption, distribution, metabolism and excretion (ADME) of administered drugs in pharmacokinetics studies. OPEN ACCESS PEER-REVIEWED*NMR can be used for the observation, quantification and kinetic characterisation of ion and smallmolecule translocation across lipid bilayers and biological membranes, including those of cells, tissues and vesicles. Solid-state NM...

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Binding of Iodide to Arthromyces ramosus Peroxidase Investigated with X-ray Crystallographic Analysis, 1H and 127I NMR Spectroscopy, and Steady-state Kinetics

Cited by 32 publications

References 52 publications

Analysis of Polypeptide Movement in the SecY Channel during SecA-mediated Protein Translocation

Analysis of Polypeptide Movement in the SecY Channel during SecA-mediated Protein Translocation

Relationships of Ligand Binding, Redox Properties, and Protonation in Coprinus cinereus Peroxidase

NMR-Active Nuclei for Biological and Biomedical Applications

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