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Ion mobility mass spectrometry uncovers the impact of the patterning of oppositely charged residues on the conformational distributions of intrinsically disordered proteins
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Figure 1b: Mass spectra of p27-C-κ14, p27-C-κ31 and p27-C-κ56 sprayed from 200 mM ammonium acetate. The κ14 and κ31 permutants display wider charge state distribution ranging from [M+6H]6+ to [M+18H]18+ whilst the κ56 in range of [M+6H]6+ to [M+13H]13+.
Figure 1c: Collision Cross Section (CCS) distributions of the each charge state of the p27-C permutants sprayed from 200 mM ammonium acetate solution.
Figure 2: Global collision cross section distributions of all three permutants sprayed from 10 mM, 100 mM or 200 mM ammonium acetate.
Figure 3: Comparison of the CCS ranges observed from experimental, computational and predicted datasets.
Figure S1: Mass spectra of p27-C permutants sprayed from 10 and 100 mM ammonium acetate solutions; p27-C-κ14 (a-b), p27-C-κ31 (c-d), p27-C-κ56 (e-f).
Figure S2: CCS distributions of each charge state of the p27-C constructs, when sprayed from 10 and 100 mM ammonium acetate; p27-C-κ14 (a, d), p27-C-κ31 (b, e), p27-C-κ56 (c, f).
Figure S3: The width of the CCS distribution obtained from MD simulations of multiple protomers of the computationally explored charge states of p27-C permutants; a) p27-C-κ14; b) p27-C-κ31; c) p27-C-κ56.
Figure S4: Collision cross section values of p27-C-κ14 protomers starting from compact and extended conformation, covering charge states [M+5H]5+ to [M+15H]15+. The overlay data represents different protomer forms of the same charge state.
Figure S5: Collision cross section values of p27-C-κ31 protomers starting from compact and extended conformation, covering charge states [M+5H]5+ to [M+15H]15+. The overlay data represents different protomer forms of the same charge state.
Figure S6: Collision cross section values of p27-C-κ14 protomers starting from compact and extended conformation, covering charge states [M+5H]5+ to [M+15H]15+. The overlay data represents different protomer forms of the same charge state.
Figure S7: Evolution of a) root mean square deviation (RMSD); b) Backbone radius of gyration (Rg); c) solvent accessible surface area (SASA) and d) collision cross sections of p27-C-κ14 permutant during the desolvation molecular dynamics.
Figure S8: Evolution of a) root mean square deviation (RMSD); b) Backbone radius of gyration (Rg); c) solvent accessible surface area (SASA) and d) collision cross sections of p27-C-κ31 permutant during the desolvation molecular dynamics.
Figure S9: Evolution of a) root mean square deviation (RMSD); b) Backbone radius of gyration (Rg); c) solvent accessible surface area (SASA) and d) collision cross sections of p27-C-κ56 permutant during the desolvation molecular dynamics.
All interactive figures were generated using ORIGAMIANALYSE available here.
Title
Ion mobility mass spectrometry uncovers the impact of the patterning of oppositely charged residues on the conformational distributions of intrinsically disordered proteins
Authors
Rebecca Beveridge1,§, Lukasz G Migas1,§, Rahul Das2, Rohit V Pappu2, Richard Kriwacki3 and Perdita E. Barran1*
1 The Michael Barber Centre for Collaborative Mass Spectrometry, The School of Chemistry, Manchester Institute for Biotechnology, University of Manchester, Manchester, UK
2 Washington University in St. Louis, Department of Biomedical Engineering and Center for Biological Systems Engineering Campus Box 1097, One Brookings Drive, St. Louis, Missouri 63130
3 Structural Biology, MS 311, Room D1024F, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-3678
* E-mail: perdita.barran@manchester.ac.uk
§ Both authors contributed equally to this work.
Abstract
The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion Mobility-Mass Spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, k14 and k56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (k31) and k14 where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, k56 with linear segregation of oppositely charged residues, leads to limited conformational heterogeneity and a narrow distribution of charged states. Molecular dynamics simulations demonstrate that the interplay between chain solvation and intra-chain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.