Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins.

Rebecca Beveridge,Rahul K Das,Lukasz G Migas,Perdita E Barran,Rohit V Pappu,Richard W Kriwacki

doi:10.1021/jacs.8b13483

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, 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 concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain 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.

Highlights

Conformational heterogeneity is a defining hallmark of intrinsically disordered proteins (IDPs).[1]
Between [M + 9H]9+ and [M + 14H]14+ the protein presents in broad conformational distributions that increase in size to ∼2500 Å2 at [M + 14H]14+, and this single conformational family is retained for the charge states to [M + 16H]16+; we propose that at this stage, the protein is present in a highly extended conformation and any addition of protons has a negligible effect on the overall dimensions
In contrast to the charge residue model (CRM), ions produced via the chain ejection model (CEM) have higher charge states. This model applies if and only if the IDPs are akin to random coils or self-avoiding walks, since IDPs have the ability to sample a broad spectrum of conformations ranging from those that are as compact as folded proteins, they are unlikely to only undergo CEM since ejection of a compact region via CEM would be unfavorable. In light of this we previously proposed that a hybrid of the CRM and the CEM will govern the generation of the intermediate charge states that are present in a multitude of conformational families.[18]

Summary

Introduction

Conformational heterogeneity is a defining hallmark of intrinsically disordered proteins (IDPs).[1]. Understanding how intrinsically disordered regions (IDRs) mediate the function of a protein requires accurate physical descriptions of their sequence-to-conformation relationships. IDPs and IDRs are often enriched in proline, glutamic acid, lysine, serine, and glutamine, yet depleted in tryptophan, tyrosine, phenylalanine, cysteine, isoleucine, leucine, and asparagine in comparison to folded, globular proteins,[8,9] and an emerging theory suggests that the context, or adaptive location of a given residue within a protein allows modulation of different functional conformational ensembles, which govern how that region will interact with a given partner.[10] One parameter to define this “context” is the net charge per residue[11]

Methods

Results

Discussion

Conclusion