scholarly journals Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and Its Domains and How This Is Modulated upon Interaction with Cdk2/cyclin A

2018 ◽  
Author(s):  
Rebecca Beveridge ◽  
Lukasz Migas ◽  
Richard Kriwacki ◽  
Perdita E. Barran
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Intrinsically Disordered Proteins ◽  
Dynamic Properties ◽  
Cyclin A ◽  
Structural Heterogeneity ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Trimeric Complex ◽  
Conformational Landscape

Intrinsically disordered proteins have been reported to undergo ‘disorder to order’ transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states.

scholarly journals Ion Mobility Mass Spectrometry Measures the Conformational Landscape of P27 and Its Domains and How This Is Modulated upon Interaction with Cdk2/cyclin A

2018 ◽  
Author(s):  
Rebecca Beveridge ◽  
Lukasz Migas ◽  
Richard Kriwacki ◽  
Perdita E. Barran
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Intrinsically Disordered Proteins ◽  
Dynamic Properties ◽  
Cyclin A ◽  
Structural Heterogeneity ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Trimeric Complex ◽  
Conformational Landscape

Intrinsically disordered proteins have been reported to undergo ‘disorder to order’ transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states.

scholarly journals Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and Its Domains and How This Is Modulated upon Interaction with Cdk2/cyclin A

2018 ◽  
Author(s):  
Rebecca Beveridge ◽  
Lukasz Migas ◽  
Richard Kriwacki ◽  
Perdita E. Barran
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Intrinsically Disordered Proteins ◽  
Dynamic Properties ◽  
Cyclin A ◽  
Structural Heterogeneity ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Trimeric Complex ◽  
Conformational Landscape

Intrinsically disordered proteins have been reported to undergo ‘disorder to order’ transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states.

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

2018 ◽  
Author(s):  
Rebecca Beveridge ◽  
Lukasz Migas ◽  
Rahul Das ◽  
Rohit Pappu ◽  
Richard Kriwacki ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Ion Mobility Mass Spectrometry ◽  
Conformational Heterogeneity ◽  
Wild Type ◽  
Intrinsically Disordered ◽  
Charged Residues ◽  
Oppositely Charged

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<sup>Kip1</sup>. 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.

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

2018 ◽  
Author(s):  
Rebecca Beveridge ◽  
Lukasz Migas ◽  
Rahul Das ◽  
Rohit Pappu ◽  
Richard Kriwacki ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Ion Mobility Mass Spectrometry ◽  
Conformational Heterogeneity ◽  
Wild Type ◽  
Intrinsically Disordered ◽  
Charged Residues ◽  
Oppositely Charged

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<sup>Kip1</sup>. 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.

Molecular simulations of protein disorderThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (2) ◽  
pp. 269-290 ◽  
Author(s):  
Sarah Rauscher ◽  
Régis Pomès
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Peer Review Process ◽  
Structural Heterogeneity ◽  
Disordered Proteins ◽  
Cellular Biology ◽  
Protein Disorder ◽  
Conformational Ensembles ◽  
Intrinsically Disordered ◽  
Simulation Techniques ◽  
Selection Of

Protein disorder is abundant in proteomes throughout all kingdoms of life and serves many biologically important roles. Disordered states of proteins are challenging to study experimentally due to their structural heterogeneity and tendency to aggregate. Computer simulations, which are not impeded by these properties, have recently emerged as a useful tool to characterize the conformational ensembles of intrinsically disordered proteins. In this review, we provide a survey of computational studies of protein disorder with an emphasis on the interdisciplinary nature of these studies. The application of simulation techniques to the study of disordered states is described in the context of experimental and bioinformatics approaches. Experimental data can be incorporated into simulations, and simulations can provide predictions for experiment. In this way, simulations have been integrated into the existing methodologies for the study of disordered state ensembles. We provide recent examples of simulations of disordered states from the literature and our own work. Throughout the review, we emphasize important predictions and biophysical understanding made possible through the use of simulations. This review is intended as both an overview and a guide for structural biologists and theoretical biophysicists seeking accurate, atomic-level descriptions of disordered state ensembles.

scholarly journals Cyclic Ion Mobility – Collision Activation Experiments Elucidate Protein Behaviour in the Gas-Phase

2020 ◽  
Author(s):  
Charles Eldrid ◽  
Jakub Ujma ◽  
Hannah Britt ◽  
Tristan Cragnolini ◽  
Symeon Kalfas ◽  
...  
Keyword(s):  
Gas Phase ◽  
Ion Mobility ◽  
Protein Unfolding ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Globular Proteins ◽  
Disordered Proteins ◽  
Mass Selection ◽  
Intrinsically Disordered ◽  
Partially Folded States

<i>Elucidating the properties of intrinsically disordered proteins (IDPs) and unfolded and partially folded states of globular proteins is challenging owing to their heterogeneous and dynamic nature. Protein unfolding and misfolding is a key feature of a broad range of debilitating diseases, whilst the conformational propensities of intrinsically disordered proteins can play a significant role in modulating their activity, and the properties of unfolded states of globular proteins modulates their stability and tendency to aggregate. Ion mobility-mass spectrometry (IM-MS) is a powerful method for interrogating these systems, however limits in resolution and the difficulty in probing the energetics of interconversions amongst heterogeneous ensembles are major issues. Herein, using a quadrupole/cyclic-IM/ time-of-flight MS instrument, we show how the combination of precursor mass selection, mobility selection (IM<sup>n</sup>) and collisional activation (CA) allows the elucidation of complicated gas-phase dynamic behavior. The methodology employed is general and is demonstrated using a classic model globular protein, cytochrome C, and an aggregation-prone IDP, amylin. CA allows investigations of protein conformational dynamics and unfolding in the gas-phase for heterogeneous mixtures, whilst the additional precursor mass selection capability provides high resolution and selectivity, facilitating more in-depth investigation. Understanding protein dynamics in the gas-phase will allow greater insight into protein behaviour and allow application of gas-phase techniques to clinically relevant systems. </i>

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