An integrative approach combining ion mobility mass spectrometry, X-ray crystallography and NMR spectroscopy to study the conformational dynamics of ?1-antitrypsin upon ligand binding
Abstract
Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whilst ion mobility (IM)-MS can report on conformational behaviour of specific states. We used IM-MS to study a conformationally labile protein (?1-antitrypsin) that undergoes pathological polymerisation in the context of point mutations. The folded, native state of the Z variant remains highly polymerogenic in physiological conditions, despite only minor thermodynamic destabilisation relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z ?1-antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behaviour in detail by studying the effects of peptide binding on ?1-antitrypsin conformation and dynamics. IM-MS is therefore an ideal platform for the screening of compounds that result in therapeutically-beneficial kinetic stabilisation of native ?1-antitrypsin. Our findings are confirmed with high resolution X-ray crystallographic and NMR spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue specific level. IM-MS methods therefore have great potential for further study of biologically-relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterisation of ligand interactions of therapeutic interest. This article is protected by copyright. All rights reserved.
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