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Bauman A.,Johannes Gutenberg University Mainz | Bauman A.,University of Basel | Piel M.,Johannes Gutenberg University Mainz | Hohnemann S.,Johannes Gutenberg University Mainz | And 6 more authors.
Journal of Labelled Compounds and Radiopharmaceuticals

The N-methyl- d-aspartate (NMDA) receptor as a type of ionotropic glutamatergic receptors is essential for physiological processes such as learning, memory and synaptic plasticity. A glutamate-induced overactivation of these receptors, accompanied by increased intracellular calcium concentration, causes cell injury and leads to a large number of acute or chronic neurological disorders, such as stroke, trauma, Parkinson's disease and Alzheimer's disease. In an attempt to visualise the glutamatergic neurotransmission in vivo with positron emission tomography, novel fluoroethoxy- and methoxy-substituted reference compounds based on the lead structure of a hydantoin-substituted indole-2-carboxylic acid were synthesised. The affinities towards the glycine binding site of the NMDA receptor showed K i values between 322 and 11 nM and the lipophilicities ranged from logD values of 1.51 to 2.53. On the basis of these results, precursor compounds were synthesised containing a phenolic hydroxy moiety to obtain the radiolabelled ligands through an alkylation reaction. Radiosynthesis was achieved by labelling the precursor ethyl 4,6-dichloro-3-((3-(4-hydroxyphenyl)-2,4-dioxoimidazolidin-1-yl)methyl)- indole-2-carboxylate with 2-[ 18F]fluoroethyl tosylate or [ 11C]methyl iodide and subsequent cleavage of the ethyl ester moiety. This gave the final products in overall decay-corrected radiochemical yields of 5-7% and 6-9% and specific activities of 24-67 GBq/μmol and 8-26 GBq/μmol, respectively. Copyright © 2011 John Wiley & Sons, Ltd. Source

Zoghbi M.E.,Center for Membrane Protein Research | Altenberg G.A.,Center for Membrane Protein Research
Journal of Biological Chemistry

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, formingadimerinahead-to-tail arrangement, with two nucleotides "sandwiched" at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysisatoneorboth nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Cooper R.S.,Center for Membrane Protein Research | Altenberg G.A.,Center for Membrane Protein Research
Journal of Biological Chemistry

Background: In ATP-binding cassette proteins, ATP binding produces association of the two nucleotide-binding domains (NBDs), but the molecular mechanism is unknown. Results: The NBDs separate following ATP hydrolysis. Conclusion: NBD dimers dissociate during the hydrolysis cycle supporting monomer/dimer models of operation. Significance: Knowledge of the molecular mechanism of hydrolysis will help us understand how ATP-binding cassette proteins work. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Niwayama S.,Texas Tech University | Kassar A.S.,Center for Membrane Protein Research | Zhao T.,Texas Tech University | Sutton R.B.,Center for Membrane Protein Research | Altenberg G.A.,Center for Membrane Protein Research

Pyrene-containing compounds are commonly used in a number of fluorescence-based applications because they can form excited-state dimers (excimers) by stacking interaction between excited-state and ground-state monomers. Their usefulness arises from the facts that excimer formation requires close proximity between the pyrenes and that the excimer emission spectrum is very different from that of the monomers. One of many applications is to assess proximity between specific sites of macromolecules labeled with pyrenes. This has been done using pyrene maleimide, a reagent that reacts with reduced thiols of cysteines, but its use for structural studies of proteins has been rather limited. This is because the introduction of two cysteines at sufficiently close distance from each other to obtain excimer fluorescence upon labeling with pyrene maleimide requires detailed knowledge of the protein structure or extensive site-directed mutagenesis trials. We synthesized and tested a new compound with a 4-carbon methylene linker placed between the maleimide and the pyrene (pyrene-4-maleimide), with the aim of increasing the sampling distance for excimer formation and making the use of excimer fluorescence simpler and more widespread. We tested the new compound on thiol-modified oligonucleotides and showed that it can detect proximity between thiols beyond the reach of pyrene maleimide. Based on its spectroscopic and chemical properties, we suggest that pyrene-4-maleimide is an excellent probe to assess proximities between cysteines in proteins and thiols in other macromolecules, as well as to follow conformational changes. © 2011 Niwayama et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source

Zoghbi M.E.,Center for Membrane Protein Research | Krishnan S.,Center for Membrane Protein Research | Altenberg G.A.,Center for Membrane Protein Research
Journal of Biological Chemistry

ATP-binding cassette (ABC) proteins have two nucleotide-binding domains (NBDs) that work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. In particular, it is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or opening of the dimers, with the NBDs remaining in contact during the hydrolysis cycle. We studied a prototypical ABC NBD, the Methanococcus jannaschii MJ0796, using spectroscopic techniques. We show that fluorescence from a tryptophan positioned at the dimer interface and luminescence resonance energy transfer between probes reacted with single-cysteine mutants can be used to follow NBD association/dissociation in real time. The intermonomer distances calculated from luminescence resonance energy transfer data indicate that the NBDs separate completely following ATP hydrolysis, instead of opening. The results support ABC protein NBD association/dissociation, as opposed to constant-contact models. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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