title: Insights into the conformations of three structurally diverse proteins: cytochrome c, p53, and MDM2, provided by variable-temperature ion mobility mass spectrometry.
abstract: Thermally induced conformational transitions of three proteins of increasing intrinsic disorder-cytochrome c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)-have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome c, dimers of significant intensity are also observed. Cytochrome c [M + 5H](5+) ions present in one conformer of CCS ∼1200 Å(2), undergoing compaction in line with the reported Tmelt = 360.15 K before slight unfolding at 571 K. The more extended [M + 7H](7+) cytochrome c monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H](11+) dimer presents as two conformers, which undergo slight structural compaction or annealing before dissociation. p53 DBD follows a trend of structural collapse before an increase in the observed collision cross section (CCS), akin to that observed for cytochrome c but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS ∼1750 Å(2) at 365 K, in line with the low solution Tmelt = 315-317 K. The protein then extends to produce either a broad unresolved CCS distribution or, for z > 9, two conformers. NT-MDM2 exhibits a greater number of structural rearrangements, displaying charge-state-dependent unfolding pathways. DT-IM-MS experiments at 200 K resolve multiple conformers. Low charge state species of NT-MDM2 present as a single compact conformational family centered on CCS ∼1250 Å(2) at 300 K. This undergoes conformational tightening in line with the solution Tmelt = 348 K before unfolding at the highest temperatures. The more extended charge states present in two or more conformers at room temperature, undergoing thermally induced unfolding before significant structural collapse or annealing at high temperatures. Variable-temperature IM-MS is here shown to be an exciting approach to discern protein unfolding pathways for conformationally diverse proteins.
authors: †Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, United Kingdom.
authors: Eleanor R
authors: ER
authors: Dickinson
authors: ‡School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom.
authors: Ewa
authors: E
authors: Jurneczko
authors: †Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, United Kingdom.
authors: Kamila J
authors: KJ
authors: Pacholarz
authors: ‡School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom.
authors: David J
authors: DJ
authors: Clarke
authors: ‡School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom.
authors: Matthew
authors: M
authors: Reeves
authors: §Institute of Genetics and Molecular Medicine, CRUK Cancer Research Centre, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom.
authors: Kathryn L
authors: KL
authors: Ball
authors: §Institute of Genetics and Molecular Medicine, CRUK Cancer Research Centre, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom.
authors: Ted
authors: T
authors: Hupp
authors: ‡School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom.
authors: Dominic
authors: D
authors: Campopiano
authors: ∥School of Biomedical Science, Institute of Pharmaceutical Sciences, King's College London, 150 Stamford Street, London SE1 9NH, United Kingdom.
authors: Penka V
authors: PV
authors: Nikolova
authors: †Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, United Kingdom.
authors: Perdita E
authors: PE
authors: Barran
mesh_terms: D000818:Animals; D045304:Cytochromes c / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry*; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D016159:Tumor Suppressor Protein p53; D045304:Cytochromes c; D004247:DNA; D051736:Proto-Oncogene Proteins c-mdm2