{"id":340,"date":"2015-10-08T14:13:35","date_gmt":"2015-10-08T14:13:35","guid":{"rendered":"https:\/\/www.mbc.manchester.ac.uk\/barrangroup\/?p=340"},"modified":"2018-10-15T12:57:23","modified_gmt":"2018-10-15T12:57:23","slug":"insights-into-the-conformations-of-three-structurally-diverse-proteins-cytochrome-c-p53-and-mdm2-provided-by-variable-temperature-ion-mobility-mass-spectrometry","status":"publish","type":"post","link":"https:\/\/www.mbc.manchester.ac.uk\/barrangroup\/2015\/10\/08\/insights-into-the-conformations-of-three-structurally-diverse-proteins-cytochrome-c-p53-and-mdm2-provided-by-variable-temperature-ion-mobility-mass-spectrometry\/","title":{"rendered":"Insights into the Conformations of Three Structurally Diverse Proteins: Cytochrome c, p53, and MDM2, Provided by Variable-Temperature Ion Mobility Mass Spectrometry"},"content":{"rendered":"

Thermally induced conformational transitions of three proteins of increasing intrinsic disorder\u2014cytochrome\u00a0c<\/em>, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)\u2014have 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\u00a0c<\/em>, dimers of significant intensity are also observed. Cytochrome\u00a0c<\/em>\u00a0[M + 5H]5+<\/sup>\u00a0ions present in one conformer of CCS \u223c1200 \u00c52<\/sup>, undergoing compaction in line with the reported\u00a0T<\/em>melt<\/sub>\u00a0= 360.15 K before slight unfolding at 571 K. The more extended [M + 7H]7+<\/sup>\u00a0cytochrome\u00a0c<\/em>\u00a0monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H]11+<\/sup>\u00a0dimer 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\u00a0c<\/em>but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS \u223c1750 \u00c52<\/sup>\u00a0at 365 K, in line with the low solution\u00a0T<\/em>melt<\/sub>\u00a0= 315\u2013317 K.<\/p>\n

\"ellie1_pap2\"<\/p>\n

The protein then extends to produce either a broad unresolved CCS distribution or, for\u00a0z<\/em>\u00a0> 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 \u223c1250 \u00c52<\/sup>\u00a0at 300 K. This undergoes conformational tightening in line with the solution\u00a0T<\/em>melt<\/sub>\u00a0= 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.<\/p>\n

 <\/p>\n

Insights into the Conformations of Three Structurally Diverse Proteins: Cytochrome c, p53, and MDM2, Provided by Variable-Temperature Ion Mobility Mass Spectrometry<\/p>\n

Eleanor R. Dickinson, Ewa Jurneczko, Kamila J. Pacholarz, David J. Clarke, Matthew Reeves, Kathryn L. Ball, Ted Hupp, Dominic Campopiano, Penka V. Nikolova, and Perdita E. Barran<\/p>\n

Anal. Chem.<\/em>,\u00a02015<\/strong>,\u00a087<\/em>\u00a0(6), pp 3231\u20133238<\/p>\n

DOI:\u00a0<\/strong>10.1021\/ac503720v<\/a><\/p><\/blockquote>\n","protected":false},"excerpt":{"rendered":"

Thermally induced conformational transitions of three proteins of increasing intrinsic disorder\u2014cytochrome\u00a0c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)\u2014have 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 … <\/p>\n

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