IonMobility Mass Spectrometry Uncovers the Impactof the Patterning of Oppositely Charged Residues on the ConformationalDistributions of Intrinsically Disordered Proteins

摘要

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 p27^Kip1. 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, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordantwith high degrees of conformational heterogeneity in solution. Bycontrast, κ56 with linear segregation of oppositely chargedresidues leads to limited conformational heterogeneity and a narrowdistribution of charged states. Gas-phase molecular dynamics simulationsdemonstrate that the interplay between chain solvation and intrachaininteractions (self-solvation) leads to conformational distributionsthat are modulated by salt concentration, with the wild-type sequenceshowing the most sensitivity to changes in salt concentration. Theseresults suggest that the charge patterning within the wild-type p27IDR may be optimized to sample both highly solvated and self-solvatedconformational states.