1. Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations
- Author
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Eric Ennifar, Gilles Guichard, Philippe Wolff, Dominique Burnouf, Guillaume Bec, Philippe Dumas, Cyrielle Da Veiga, Université Lumière - Lyon 2 (UL2), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Immunologie et chimie thérapeutiques (ICT), Cancéropôle du Grand Est-Centre National de la Recherche Scientifique (CNRS), Laboratoire LePont, and Université de Toulon et du Var (UTV)
- Subjects
0301 basic medicine ,DNA-polymerase processivity rings ,Electrospray ionization ,[SDV]Life Sciences [q-bio] ,Analytical chemistry ,Peptide ,010402 general chemistry ,01 natural sciences ,Dissociation (chemistry) ,03 medical and health sciences ,Structural Biology ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Binding site ,Spectroscopy ,ComputingMilieux_MISCELLANEOUS ,chemistry.chemical_classification ,ESI-MS ,Isothermal titration calorimetry ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,Processivity ,ESI mass spectrometry ,ITC ,0104 chemical sciences ,Kd determination ,Crystallography ,030104 developmental biology ,chemistry ,Titration ,Research Article - 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 Kd values (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 Abstractᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-016-1534-6) contains supplementary material, which is available to authorized users.
- Published
- 2017
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