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 ᅟ
Highlights
Mass spectrometry using electrospray ionization (ESI-MS) in native conditions has been pioneered in 1991 by two Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.P
The Isothermal titration calorimetry (ITC) titration curves and their fits with the same model as for ESI-MS data processing are shown in Supplementary Figure S8
The comparison of the KdðESI–MSÞ and available Kd(ITC) values is shown in Figure 6
Summary
Mass spectrometry using electrospray ionization (ESI-MS) in native conditions has been pioneered in 1991 by two 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. The extrapolation of Kd at 22.5 °C was always performed from a temperature close to 22.5 °C The latter simple processing method did not allow us to obtain a correct fit of several ITC data and, we attempted to use a more complex (but classic) model by considering a competition between two modes of binding on the same site. This competition model requires determining a fixed fraction f1 of the protein monomers binding their ligand according to a dissociation constant Kd1 and an enthalpy variation ΔH1, and a fixed fraction f2 = 1 − f1 binding their ligand according to a dissociation constant Kd2 and an enthalpy variation ΔH2 (we consider Kd2 < Kd1) Since it appeared clearly with the ESI-MS data from βE.coli/P6 (Figure 2) that a third peptide (and marginally a fourth peptide) could bind the β -ring, it was attempted to see whether this weak association was detectable in the ITC data from βE.coli/P6. All data processing was performed with our own methods programmed with Mathematica (Wolfram Research)
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