title: Determinants of the pKa values of ionizable residues in an intrinsically disordered protein.
abstract: Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes; in humans, they are often associated with diseases. The protein NUPR1 is a multifunctional IDP involved in the development and progression of pancreatic cancer; therefore, it constitutes a target for drug design. In an effort to contribute to the understanding of the conformational features of NUPR1 and to provide clues on amino acid interactions in disordered states of proteins, we measured the pKa values of all its acidic groups (aspartic and glutamic residues, and backbone C terminus) by using NMR spectroscopy at low (100 mM) and high (500 mM) NaCl concentration. At low ionic strength, the pKa values were similar to those reported for random-coil models, except for Glu18 and Asp19, suggesting electrostatic interactions around these residues. Molecular modelling and simulation indicate an additional, significant role of nearby proline residues in determining the polypeptide conformational features and water accessibility in the region around Glu18, modulating the titration properties of these amino acids. In the other acidic residues of NUPR1, the small deviations of pKa values (compared to those expected for a random-coil) are likely due to electrostatic interactions with charged adjacent residues, which should be reduced at high NaCl concentrations. In fact, at high ionic strength, the pKa values of the aspartic residues were similar to those in a random coil, but there were still small differences for those of glutamic acids, probably due to hydrogen-bond formation. The overall findings suggest that local interactions and hydrophobic effects play a major role in determining the electrostatic features of NUPR1, whereas long-range charge contributions appear to be of lesser importance.
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain; Biocomputation and Complex Systems Physics Institute, 50009 Zaragoza, Spain. Electronic address: jlneira@umh.es.
authors.forename: José L
authors.initials: JL
authors.lastname: Neira
authors.affiliation: CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of Calabria, 87036 Rende, Italy. Electronic address: bruno.rizzuti@cnr.it.
authors.forename: Bruno
authors.initials: B
authors.lastname: Rizzuti
authors.affiliation: Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France.
authors.forename: Juan L
authors.initials: JL
authors.lastname: Iovanna
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry*; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D012965:Sodium Chloride / Q000737:chemistry; D055672:Static Electricity
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D009363:Neoplasm Proteins; D012965:Sodium Chloride; C491643:NUPR1 protein, human
keywords: Electrostatic interactions; Molecular dynamics; NMR; Random-coil; Titration; pK(a) values