title: Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random-coil-like pKavalues.
abstract: The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65-residue-long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pKavalues of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C-terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pKavalues. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random-coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N-terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.
authors.affiliation: Molecular Biophysics Laboratory, Department of Physics, University of Calabria, Rende, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Cozza
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Alicante, Spain.|Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Unidad Asociada IQFR-CSIC-BIFI, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: José L
authors.initials: JL
authors.lastname: Neira
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Seville, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Florencio
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Seville, Spain.
authors.forename: M Isabel
authors.initials: MI
authors.lastname: Muro-Pastor
authors.affiliation: CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of Calabria, Rende, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Rizzuti
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D064267:Intrinsically Disordered Proteins / Q000737:chemistry*; D000072417:Protein Domains; D046939:Synechocystis / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D064267:Intrinsically Disordered Proteins; D005974:Glutamate-Ammonia Ligase
keywords: electrostatics; intrinsically disordered proteins; molecular dynamics; nuclear magnetic resonance; pKa values; titration