Unusual ECD fragmentation attributed to gas-phase helix formation in a conformationally dynamic peptide

Kalapothakis, Jason M. D.; Berezovskaya, Yana; Zampronio, Cleidiane G.; Faull, Peter; Barran, Perdita E.; Cooper, Helen J.

doi:10.1039/c3cc46356g

Cited by 17 publications

(13 citation statements)

References 21 publications

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“…This was interpreted as evidence for ECD data reflecting protein structure in the gas phase, in that the observation of separated c and z • fragments indicates the lack of noncovalent bonding between each other [ 9 ]. Ever since, the unusual capability of ECD to break covalent N–Cα bonds while maintaining noncovalent bonds that account for the higher order structure of gaseous ions has been used to obtain information about peptide or protein structure in the absence of solvent [ 7 , 9 – 25 ]. More recently, this approach has been extended to the study of noncovalently bound protein complexes [ 18 , 26 – 31 ].…”

Section: Historymentioning

confidence: 99%

Probing Protein Structure and Folding in the Gas Phase by Electron Capture Dissociation

Schennach

Breuker

2015

J. Am. Soc. Mass Spectrom.

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The established methods for the study of atom-detailed protein structure in the condensed phases, X-ray crystallography and nuclear magnetic resonance spectroscopy, have recently been complemented by new techniques by which nearly or fully desolvated protein structures are probed in gas-phase experiments. Electron capture dissociation (ECD) is unique among these as it provides residue-specific, although indirect, structural information. In this Critical Insight article, we discuss the development of ECD for the structural probing of gaseous protein ions, its potential, and limitations.Graphical Abstractᅟ

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Section: Historymentioning

confidence: 99%

Probing Protein Structure and Folding in the Gas Phase by Electron Capture Dissociation

Schennach

Breuker

2015

J. Am. Soc. Mass Spectrom.

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“…Ultraviolet photodissociation has recently also been combined with IM, showing that different conformations of ubiquitin lead to different fragmentation patterns . Electron‐based methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) cleave the backbone of a protein or peptide while leaving labile PTMs and noncovalent interactions largely intact . It should be noted that in these experiments, the gain of information about the higher‐order structure of the native protein typically comes at the cost of a reduced fragmentation efficiency and sequence coverage, compared to ECD or ETD of denatured proteins .…”

Section: Introductionmentioning

confidence: 99%

Electron transfer dissociation provides higher‐order structural information of native and partially unfolded protein complexes

Lermyte

Sobott

2015

Proteomics

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Top-down sequencing approaches are becoming ever more popular for protein characterization, due to the ability to distinguish and characterize different protein isoforms. Under non-denaturing conditions, electron transfer dissociation (ETD) can furthermore provide important information on the exposed surface of proteins or complexes, thereby contributing to the characterization of their higher-order structure. Here, we investigate this approach using top-down ETD of tetrameric hemoglobin, concanavalin A, and alcohol dehydrogenase combined with ion mobility (IM) on a commercially available quadrupole/ion mobility/time-of-flight instrument (Waters Synapt G2). By applying supplemental activation in the transfer cell (post-IM), we release ETD fragments and attain good sequence coverage in the exposed terminal regions of the protein. We investigate the correlation between observed sites of fragmentation with regions of solvent accessibility, as derived from the crystal structure. Ion acceleration prior to ETD is also used to cause collision-induced unfolding (CIU) of the complexes without monomer ejection, as evidenced by the IM profiles. These partially unfolded tetramers show efficient fragmentation in some regions which are not sequenced under more gentle MS conditions. We show that by increasing CIU in small increments and monitoring the changes in the fragmentation pattern, it is possible to follow the initial steps of gas-phase protein unfolding. Fragments from partially unfolded protein complexes are released immediately after electron transfer, prior to IM (they do not share the drift time of their precursor), and observed without the need for supplemental activation. This is further evidence that the higher-order structure in these protein regions has been disrupted.

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“…In such a conformation protonated residues would be held away from each other, minimizing coulombic repulsion, which would result in no or little increase in CCS with respect to increased protonation. [11,12] This would indicate that the lower charge states are more charge solvated forms, which is supported by the substantial adductation of the protein by sodium and potassium for z=5-7, and the fact that a substantial portion of the measured conformational spread comes from charges states that fall below the limit predicted for compact/folded states. [7] The native mass spectrum of p27-FL (Figure 2d) exhibits a wide CSD from [M+9H] 9+ to [M+33H] 33+ ; the multimodal nature of the CSD reveals the coexistence of multiple distinct conformational families upon desolvation.…”

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confidence: 91%

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

Beveridge¹,

Migas

Kriwacki³

et al. 2018

Preprint

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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.

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Unusual ECD fragmentation attributed to gas-phase helix formation in a conformationally dynamic peptide

Cited by 17 publications

References 21 publications

Probing Protein Structure and Folding in the Gas Phase by Electron Capture Dissociation

Probing Protein Structure and Folding in the Gas Phase by Electron Capture Dissociation

Electron transfer dissociation provides higher‐order structural information of native and partially unfolded protein complexes

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

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