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.

Molecular Insights into the Thermal Stability of mAbs with Variable‐Temperature Ion‐Mobility Mass Spectrometry

“…[16,17] Variationsi ns tability between antibodies of the same subclass come from differences in the sequence of the variable domain. [22][23][24] VT-IM-MS measurementso nt hree intact antibodies with the same antigen binding specificity (IgG1, IgG4 and ah ybrid IgG4 with inserted IgG1 upper and core hinge, denoted IgG4DIgG1-hinge) as well as two Fc-hingef ragmentsa re reported here. [7,11] In this work, we exploit the hybrid technique of ion mobility mass spectrometry (IM-MS), which provides shape-defining parameters to terms of collisionc rosssections ( DT CCS He )a nd facilitates the study of the higher-order structure of proteins such as mAbs or their derivatives.…”

Aggregation of Chameleon Peptides: Implications of α-Helicity in Fibril Formation

“…This is important in order to retain solution-like structures for cross section determinantion. 58–62 …”

Charge Mediated Compaction and Rearrangement of Gas-Phase Proteins: A Case Study Considering Two Proteins at Opposing Ends of the Structure-Disorder Continuum

“…When sprayed from aqueous salty solutions, IDPs have wide multimodal charge state distributions [53, 54]. Electrospray mass spectrometry coupled to ion mobility is gaining popularity as a method to study these proteins [24, 55, 56]. Thus the questions we sought to answer in this study are as followed.…”