Charge Movement and Structural Changes in the Gas-Phase Unfolding of Multimeric Protein Complexes Captured by Native Top-Down Mass Spectrometry

Zhou, Mowei; Liu, Weijing; Shaw, Jared B.

doi:10.1021/acs.analchem.9b03469

Title: Charge Movement and Structural Changes in the Gas-Phase Unfolding of Multimeric Protein Complexes Captured by Native Top-Down Mass Spectrometry

Journal Article · Mon Dec 23 00:00:00 EST 2019 · Analytical Chemistry

DOI:https://doi.org/10.1021/acs.analchem.9b03469· OSTI ID:1597653

^[1]; Liu, Weijing ^[1];

^[1]

Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

The extent to which noncovalent protein complexes retain native structure in the gas phase is highly dependent on experimental conditions. Energetic collisions with background gas can cause structural changes ranging from unfolding to subunit dissociation. Additionally, recent studies have highlighted the role of charge in such structural changes, but the mechanism is not completely understood. In this study, native top down (native TD) mass spectrometry was used to probe gas-phase structural changes of alcohol dehydrogenase (ADH, 4mer) under varying degrees of in-source activation. Changes in covalent backbone fragments produced by electron capture dissociation (ECD) or 193 nm ultraviolet photodissociation (UVPD) were correlated to structural changes of the ADH 4mer. ECD fragments indicated unfolding started at the N-terminus, and the charge states of UVPD fragments enabled monitoring of charge migration to the unfolded regions. Interestingly, UVPD fragments also indicated that the charge at the “unfolding” N-terminus of ADH decreased at high in-source activation energies after the initial increase. We proposed a possible “refolding-after-unfolding” mechanism, as further supported by monitoring hydrogen elimination from radical a-ions produced by UVPD at the N-terminus of ADH. However, “refolding-after-unfolding” with increasing in-source activation was not observed for charge-reduced ADH, which likely adopted compact structures that are resistant to both charge migration and unfolding. Combined, these results support a charge-directed unfolding mechanism for protein complexes. Overall, an experimental framework was outlined for utilizing native TD to generate structure-informative mass spectral signatures for protein complexes that complement other structure characterization techniques, such as ion mobility spectrometry and computational modeling.

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Research Organization:: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1597653

Report Number(s):: PNNL-SA-145158

Journal Information:: Analytical Chemistry, Vol. 92, Issue 2; ISSN 0003-2700

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 24 works

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Title: Charge Movement and Structural Changes in the Gas-Phase Unfolding of Multimeric Protein Complexes Captured by Native Top-Down Mass Spectrometry

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