Characterization of the T4 gp32–ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry

Blevins, Molly S.; Walker, Jada N; Schaub, Jeffrey M.; Finkelstein, Ilya J.; Brodbelt, Jennifer S.

doi:10.1039/d1sc02861h

Cited by 5 publications

(4 citation statements)

References 86 publications

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“…Native MS has been used to reveal the structure, affinity and stoichiometric ratios of noncovalent protein‐DNA/RNA complexes (Largy et al, 2021). Here we will focus on recent nTDMS studies in this field (Blevins et al, 2021; Gordiyenko et al, 2008; Leopold et al, 2005; Ma et al, 2014; Schneeberger et al, 2017; Schneeberger et al, 2020; Vusurovic & Breuker, 2019; Vusurovic et al, 2017; J. Zhang et al, 2017).…”

Section: Applicationsmentioning

confidence: 99%

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

Liu

Xia

2022

Mass Spectrometry Reviews

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Progress in structural biology research has led to a high demand for powerful and yet complementary analytical tools for structural characterization of proteins and protein complexes. This demand has significantly increased interest in native mass spectrometry (nMS), particularly native top‐down mass spectrometry (nTDMS) in the past decade. This review highlights recent advances in nTDMS for structural research of biological assemblies, with a particular focus on the extra multi‐layers of information enabled by TDMS. We include a short introduction of sample preparation and ionization to nMS, tandem fragmentation techniques as well as mass analyzers and software/analysis pipelines used for nTDMS. We highlight unique structural information offered by nTDMS and examples of its broad range of applications in proteins, protein‐ligand interactions (metal, cofactor/drug, DNA/RNA, and protein), therapeutic antibodies and antigen‐antibody complexes, membrane proteins, macromolecular machineries (ribosome, nucleosome, proteosome, and viruses), to endogenous protein complexes. The challenges, potential, along with perspectives of nTDMS methods for the analysis of proteins and protein assemblies in recombinant and biological samples are discussed.

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Section: Applicationsmentioning

confidence: 99%

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

Liu

Xia

2022

Mass Spectrometry Reviews

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“…One extensively studied model for such interactions is the Gene 32 protein (gp32), the single-stranded (ss) DNA binding protein of the bacteriophage T4, a Zn 2+ metalloprotein. 48,49 It transiently and cooperatively binds to exposed sequences of ssDNA during the DNA replication process, regulating interactions between other sub-assemblies of the replication complex throughout the replication cycle. 12 gp32 is thought to be able to quickly cover those transiently single stranded regions that arise near the advancing T4 DNA replication forks and in so doing stabilizes a particular ssDNA conformation that is most appropriate to serve as a substrate for other catalytic proteins.…”

Section: Introductionmentioning

confidence: 99%

Single molecule technique unveils the role of electrostatic interactions in ssDNA–gp32 molecular complex stability

Schiopu,

Dragomir,

Asandei

2024

RSC Adv.

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“…Protein structures can be determined via various structural biology modalities, ranging from conventional, high-resolution tools such as CryoEM, − NMR, , and X-ray crystallography , to low-resolution mass-spectrometry-based methods. , Various mass spectrometric approaches, such as chemical labeling, photoaffinity labeling, and hydrogen–deuterium exchange (HDX) MS, − along with gas-phase dissociation methods including electron-capture dissociation (ECD) − and ultraviolet photodissociation (UVPD), − afford lower structural resolution but superior sensitivity compared to the atomic-resolution of CryoEM, NMR, and X-ray crystallography. , For example, top–down protein cross-linking mass spectrometry (XL-MS) enables the deduction of local secondary structures as well as general topologies of proteins via the distance constraints imposed by cross-linkers. − The protein cross-linking reaction is typically performed in solution, followed by gas-phase tandem mass spectrometry (MS n , where n > 1) fragmentation of the modified protein to localize the linker attachments. − The cross-linking reaction, however, can alternatively be performed in the gas phase via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions. − For example, the McLuckey group used ubiquitin cations and ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions to first develop a gas-phase XL-MS workflow . The reactant ions with opposite charges were mutually confined in a radio frequency (RF) ion trap to effect a cross-linking reaction.…”

Section: Introductionmentioning

confidence: 99%

Structural Elucidation of Ubiquitin via Gas-Phase Ion/Ion Cross-Linking Reactions Using Sodium-Cationized Reagents Coupled with Infrared Multiphoton Dissociation

Kang,

Mondal,

Bonney

et al. 2024

Anal. Chem.

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Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N-hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

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Characterization of the T4 gp32–ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry

Abstract: Protein-DNA interactions play crucial roles in DNA replication across all living organisms. Here, we apply a suite of mass spectrometry (MS) tools to characterize a protein-ssDNA complex, T4 gp32•ssDNA, with...

Cited by 5 publications

References 86 publications

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

Single molecule technique unveils the role of electrostatic interactions in ssDNA–gp32 molecular complex stability

Structural Elucidation of Ubiquitin via Gas-Phase Ion/Ion Cross-Linking Reactions Using Sodium-Cationized Reagents Coupled with Infrared Multiphoton Dissociation

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