On the Formation of Highly Charged Gaseous Ions from Unfolded Proteins by Electrospray Ionization

Konermann, Lars; Rodriguez, Antony D.; Liu, Jiangjiang

doi:10.1021/ac301298g

Cited by 68 publications

(92 citation statements)

References 45 publications

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“…The spectrum is shifted from the high charge state pattern characteristic of unfolded protein toward the much lower charge state distribution of the native protein, consistent with a significant compaction and reduction in surface exposure to solvent (26,27). A small population fraction with CSD similar to that of the unfolded protein is also seen but it can be noted that the populations measured in this way are greatly biased toward exaggerating the more unfolded component (26). All of these results point to a fast molecular compaction into an extended polyglobular condition.…”

Section: Significancementioning

confidence: 69%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

107

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Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.SAXS | HDX | protein collapse | denatured state ensemble F ifty years after Anfinsen's seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3-7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8-12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work...

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Section: Significancementioning

confidence: 69%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

107

View full text Add to dashboard Cite

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“…29,30 32 These relationships underpin the use of ESI-MS to characterize the conformations of proteins with more open structures, where increase charge signatures are interpreted by the associated increase in SASA; enabling charge to be used as a restraint for structural modelling 14,3124 More recently the ionization of flexible species has been described by extrusion or the Chain Ejection Mechanism (CEM); the latter of which will be referred to from hereon due to its particular relevance to the behavior of proteins. 27,28 In the CEM charge transfer drives the ejection of highly charged proteins from ESI droplets prior to the latter stages of droplet dehydration. Protein ejection confounds canonical charge-structure relations for IDPs as the z ave. of these proteins is also influenced strongly by variables extraneous to their SASA such as the size and charge of the electrospray droplet from which they emerge.…”

Section: Esi Imposes Additional Subpopulations In Idpsmentioning

confidence: 99%

“…25,26 Recent shifts in our understanding of how flexible species escape electrospray droplets during ESI also add to the complexity of describing the solution to gas-phase transfer of IDPs. 27,28 The present challenge is to understand how to reconcile these recent findings within more established frameworks describing the behavior of disordered proteins in the solvent-free environment. A reevaluation of the solution to gas-phase transfer of IDPs is required.…”

Section: Introductionmentioning

confidence: 99%

Ensemble Methods Enable a New Definition for the Solution to Gas-Phase Transfer of Intrinsically Disordered Proteins

et al. 2015

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Intrinsically disordered proteins (IDPs) are important for health and disease, yet their lack of net structure precludes an understanding of their function using classical methods. Gas-phase techniques provide a promising alternative to access information on the structure and dynamics of IDPs, but the fidelity to which these methods reflect the solution conformations of these proteins has been difficult to ascertain. Here we use state of the art ensemble techniques to investigate the solution to gas-phase transfer of a range of different IDPs. We show that IDPs undergo a vast conformational space expansion in the absence of solvent to sample a conformational space 3-5 fold broader than in solution. Moreover, we show that this process is coupled to the electrospray ionization process, which brings about the generation of additional subpopulations for these proteins not observed in solution due to competing effects on protein charge and shape. Ensemble methods have permitted a new definition of the solution to gas-phase transfer of IDPs and provide a roadmap for future investigations into flexible systems by mass spectrometry.

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“…but systematically varied k on and/or k off (covering a broad range of values while maintaining the same K a ) would allow perturbations of the equilibrium in electrospray to be captured and the droplet lifetime to be assessed. In addition to the experimental data, the progress in computational methods now allows simulations to be performed of chemical interactions in micro-and nanodroplets at an unprecedented level of detail and deep mechanistic insight into the system dynamics to be gained [44,45].…”

Section: Discussionmentioning

confidence: 99%

On the preservation of non-covalent protein complexes during electrospray ionization

Chingin

Barylyuk

Chen

2016

Phil. Trans. R. Soc. A.

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The application range of electrospray ionization mass spectrometry for the quantitative determination of stoichiometries and binding constants for non-covalent protein complexes is broadly discussed. The underlying fundamental question is whether or not the original molecular equilibrium can be preserved during the ionization process and be revealed by subsequent mass spectrometry analysis. Here, we take a new look at this question by discussing recent studies in droplet chemistry. This article is part of the themed issue ‘Quantitative mass spectrometry’.

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On the Formation of Highly Charged Gaseous Ions from Unfolded Proteins by Electrospray Ionization

Cited by 68 publications

References 45 publications

Folding of a large protein at high structural resolution

Folding of a large protein at high structural resolution

Ensemble Methods Enable a New Definition for the Solution to Gas-Phase Transfer of Intrinsically Disordered Proteins

On the preservation of non-covalent protein complexes during electrospray ionization

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