title: Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations.
abstract: We studied by native ESI-MS the binding of various DNA-polymerase-derived peptides onto DNA-polymerase processivity rings from Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. These homodimeric rings present two equivalent specific binding sites, which leads to successive formation during a titration experiment of singly- and doubly occupied rings. By using the ESI-MS free-ring spectrum as a ruler, we derived by robust linear regression the fractions of the different ring species at each step of a titration experiment. These results led to accurate Kdvalues (from 0.03 to 0.5 μM) along with the probability of peptide loss due to gas phase dissociation (GPD). We show that this good quality is due to the increased information content of a titration experiment with a homodimer. Isothermal titration calorimetry (ITC) led with the same binding model to Kd(ITC) values systematically higher than their ESI-MS counterparts and, often, to poor fit of the ITC curves. A processing with two competing modes of binding on the same site requiring determination of two (Kd, ΔH) pairs greatly improved the fits and yielded a second Kd(ITC) close to Kd(ESI-MS). The striking features are: (1) ITC detected a minor binding mode (~20%) of 'low-affinity' that did not appear with ESI-MS; (2) the simplest processing of ITC data with only one (Kd, ΔH) pair led wrongly to the Kd of the low-affinity binding mode but to the ΔH of the high-affinity binding mode. Analogous misleading results might well exist in published data based on ITC experiments. Graphical Abstract ᅟ.
authors.affiliation: Biophysics and Structural Biology Team, Unité Architecture et réactivité de l'ARN (UPR9002), Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15, rue René Descartes, F67084, Strasbourg cedex, France.|Plateforme protéomique Strasbourg-Esplanade, Institut de Biologie Moléculaire et Cellulaire du CNRS, FRC 1589, Université de Strasbourg, 15, rue René Descartes, F67084, Strasbourg cedex, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Wolff
authors.affiliation: Biophysics and Structural Biology Team, Unité Architecture et réactivité de l'ARN (UPR9002), Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15, rue René Descartes, F67084, Strasbourg cedex, France.
authors.forename: Cyrielle
authors.initials: C
authors.lastname: Da Veiga
authors.affiliation: Biophysics and Structural Biology Team, Unité Architecture et réactivité de l'ARN (UPR9002), Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15, rue René Descartes, F67084, Strasbourg cedex, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Ennifar
authors.affiliation: Biophysics and Structural Biology Team, Unité Architecture et réactivité de l'ARN (UPR9002), Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15, rue René Descartes, F67084, Strasbourg cedex, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bec
authors.affiliation: CBMN, UMR 5248, Institut Européen de Chimie et Biologie, Université de Bordeaux, 2 rue Robert Escarpit, 33607, Pessac, France.|CNRS, CBMN, UMR 5248, 33600, Pessac, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Guichard
authors.affiliation: Biophysics and Structural Biology Team, Unité Architecture et réactivité de l'ARN (UPR9002), Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15, rue René Descartes, F67084, Strasbourg cedex, France. d.burnouf@ibmc-cnrs.unistra.fr.
authors.forename: Dominique
authors.initials: D
authors.lastname: Burnouf
authors.affiliation: Biophysics and Structural Biology Team, Unité Architecture et réactivité de l'ARN (UPR9002), Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15, rue René Descartes, F67084, Strasbourg cedex, France. p.dumas@ibmc-cnrs.unistra.fr.|Institut de Biologie et Génétique Moléculaire, Université de Strasbourg, 1, rue Laurent Fries, 67400, Illkirch, France. p.dumas@ibmc-cnrs.unistra.fr.
authors.forename: Philippe
authors.identifier: 0000-0003-4115-5175
authors.initials: P
authors.lastname: Dumas
keywords: DNA-polymerase processivity rings; ESI-MS; ITC; Kd determination