Native ESI Mass Spectrometry Can Help to Avoid Wro

Authors: Philippe Wolff Cyrielle Da Veiga Eric Ennifar Guillaume Bec Gilles Guichard Dominique Burnouf Philippe Dumas

Publish Date: 2016/12/12

Volume: 28, Issue: 2, Pages: 347-357

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Abstract

We studied by native ESIMS the binding of various DNApolymerasederived peptides onto DNApolymerase 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 ESIMS freering 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 003 to 05 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 KdITC values systematically higher than their ESIMS 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 KdITC close to KdESIMS The striking features are 1 ITC detected a minor binding mode ~20 of ‘lowaffinity’ that did not appear with ESIMS 2 the simplest processing of ITC data with only one Kd ΔH pair led wrongly to the Kd of the lowaffinity binding mode but to the ΔH of the highaffinity binding mode Analogous misleading results might well exist in published data based on ITC experimentsMass spectrometry using electrospray ionization ESIMS in native conditions has been pioneered in 1991 by two almost simultaneous studies one on the observation of enzyme–substrate and enzyme–product complexes 1 and another one on the observation of the heme–globin complex in native hemoglobin 2 Since then ESIMS in native conditions has become common usage for studying noncovalent biological complexes 3 However a major problem attached to this approach is the possible partial dissociation of the complex following the transfer into the gas phase This is known as gasphase dissociation GPD first studied quantitatively in 4 This is a major practical problem because in the absence of additional information it is difficult to disentangle the amount of GPD from the determination of the dissociation constant K d characterizing the interaction This difficulty stems from the fact that by ignoring the existence of GPD an apparent K d app may often account reasonably for the experimental data from a titration experiment see section ‘To which extent can a single K d explain data affected by GPD’ in Supplementary Data for a quantitative discussion of this problem In any case an increase of GPD leads to an apparent affinity lower than the real one hence to K d app higher than K d This becomes crucial due to the socalled ‘aggregation problem’ which is an opposite effect resulting from the strengthening of nonspecific electrostatic interactions in the gas phase This was particularly clear with positively charged antibiotics interacting with RNA 5 It is then quite possible that these nonspecific and irrelevant interactions can be mistakenly considered as genuine specific interactions particularly if the latter were thought to be lowered by GPD In the field of drug development this may become a serious problem particularly when searching for a lead compound that is expected to bind poorly Several lines of investigation have been undertaken either to minimize the problem or to take it into account at the dataprocessing stage Minimization of GPD has been obtained by using additives such as imidazole or sulfur hexafluoride in the gas phase 6 7 The favorable effect preserving the integrity of the complex of interest is thought to be due to evaporative cooling of the additive evacuating extra energy from the collisions with the neutralgas molecules Another possibility is the use of infrared radiation to heat the molecular complexes in the gas phase and dissociate nonspecific interactions 8 However a sufficient difference in stability of the specific and nonspecific interactions is necessary to avoid dissociating significantly the specific interactions too Another method not for minimizing GPD but to quantify it is to introduce both the protein of interest and an unrelated protein as a reporter for aggregation 9 10 11 The latter method was thought to be misleading for aggregation resulting from strong electrostatic interactions and was thus modified by using a reference cognate ligand competing with a putative ligand for the same site 12 Finally a processing method taking into account directly the amount of GPD in conjunction with a micro gel filtration step to remove excess of ligand was proposed 13Here we show that with a dimeric protein with two equivalent and independent binding sites the previously mentioned problems resulting from the correlation between the K d and the amount of GPD are considerably minimized for two reasons First instead of only one independent titration curve to be explained fraction of complex versus concentration of the ligand there are now two independent experimental curves to be explained fractions of singly and doubly occupied dimers versus concentration of the ligand which represents an important increase of experimental information Furthermore there are no additional free parameters to be adjusted to account for this 2fold amount of information As a consequence contrary to the previous situation it is now mathematically impossible to obtain a single K d app accounting for the two independent experimental curves if one ignores the existence of GPD In fact it may be shown that there is a mathematical solution for K d app explaining the evolution of the fraction of singly occupied dimers but it often leads to absurd negative values and there is no solution at all explaining the evolution of the fraction of doubly occupied dimers As a result the important correlation between K d and GPD with a monomeric macromolecule disappears completely with a dimer having two identical and independent binding sites We show in the following that not only the K d for the specific protein/ligand interaction and the amount of GPD but also in one case a K d like value for nonspecific aggregation could be determined accurately Importantly this was achieved without recourse to additional experimental steps such as the use of 1 gas additives 2 a reference protein or ligand and/or 3 a chromatographic step prior to injection in the instrumentIllustration of the ring species R 0 R 1 and R 2 Structure of the Ecoli processivity factor 13 Each specificallybound peptide is represented as a sphere at the position known from Xray crystallography 16 17 18 Nonspecific binding sites for a third peptide discussed in the following is are not knownThe three dnaN bacterial genes were cloned into pET15b plasmid Invitrogen Carlsbad CA USA using standard protocols The resulting Ntagged protein was expressed in BL21 E coli cells after IPTG induction 01 mM at 28 °C The β protein fractions were first enriched on a NiNTA column eluted with a histidine step 300 mM and further purified on a MonoQ column in buffer containing 20 mM Tris HCl pH 75 05 mM EDTA and 10 glycerol using a gradient from 0 to 05 M NaCl The purified proteins were dialyzed and concentrated around 300 μM against buffer containing 20 mM Tris HCl pH 75 05 mM EDTA and 10 glycerol

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