Mass spectrometry beyond the native state

Chandler, Shane A.; Benesch, Justin L. P.

doi:10.1016/j.cbpa.2017.11.019

Cited by 48 publications

(58 citation statements)

References 79 publications

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“…Detailed studies of polyprotein processing and complex formation require a method that follows these dynamic processes at a molecular level. Native mass spectrometry (MS) probes and detects protein complexes in protein mixtures, while keeping their non-covalent interactions intact through nano-electrospray ionization (nanoESI) [30,31]. Furthermore, collision-induced dissociation (CID) enables the stoichiometry and topology of natively sprayed protein complexes to be deciphered, by dissociating non-covalent interactions in vacuo.…”

Section: Introductionmentioning

confidence: 99%

Processing of the SARS-CoV pp1a/ab nsp7–10 region

Krichel

Falke

Hilgenfeld

et al. 2020

Biochemical Journal

105

119

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Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/ transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease M pro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

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Section: Introductionmentioning

confidence: 99%

Processing of the SARS-CoV pp1a/ab nsp7–10 region

Krichel

Falke

Hilgenfeld

et al. 2020

Biochemical Journal

105

119

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“…We mimicked mechanical strain on FLNC by performing experiments in which we unfolded the protein in the gas phase, along a pathway defined by the Coulombic repulsion between mobile charges (66). While the free energies of unfolding transitions can be influenced by the lack of solvent in these experiments (85, 86), electric field-induced protein mechanics have been shown to reflect functional trajectories (87). Our experiments revealed that FLNC d18-21 adopts a compact state, meaning that multiple inter-domain interactions are likely.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of HspB1 regulates its mechanosensitive molecular chaperone interaction with native filamin C

Collier

Alderson

et al. 2018

Preprint

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Small heat-shock proteins (sHsps; HspBs) are molecular chaperones involved in the cellular stress response and a range of basal functions. Despite a multitude of targets, sHsp interactions are not well understood due their heterogeneous structures and weak binding affinities. The most widely expressed human sHsp, HspB1, is prevalent in striated muscle, where the actin cross-linker filamin C (FLNC, γ-filamin, ABP-L) is a putative binding partner. Musculoskeletal HspB1 is phosphorylated in response to a variety of cues, including mechanical stress, which promotes oligomer disassembly and association with myoarchitectural elements. Here, we report the up-regulation and interaction of both proteins in the hearts of a mouse model of heart failure, with HspB1 being phosphorylated and FLNC increasingly associated with the sarcomeric Z-disc. We used a combination of structural approaches to reveal that phosphorylation of HspB1 results in increased availability of the residues surrounding the phosphosite, facilitating their interaction with folded FLNC domains equivalent to a force-sensing region in the paralog filamin A. By employing native mass spectrometry, we show that domains 18 to 21 of FLNC are extensible under conditions mimicking force, with phosphorylated HspB1 stabilising an intermediate from further unfolding. These findings report on conformations accessible during the cycles of mechanical extension central to filamin function, and are consistent with an interaction between the chaperone and a native target that is strengthened upon the application of force. This may represent a new mode of molecular chaperone activity, allowing HspB1 to protect FLNC from over-extension during mechanical stress.

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“…These procedures have been determined previously not to yield any detectable isomerization in control experiments. 31 Peptide and Radical Precursor Synthesis--All synthetic peptides were synthesized manually using standard FMOC SPPS procedures with Wang resins, or Rink Amide resins as the solid support (29). N-hydroxysuccinimide (NHS) activated iodobenzoyl esters were synthesized by following a previous procedure.…”

Section: Methodsmentioning

confidence: 99%

“…2 Here we have identified four age-related isomerization sites in αA and αB ( αB Ser59, αA Ser162, αB Asp62, αB Asp109) that reside in regions critical for oligomerization, and demonstrate how key structural interactions are disrupted by these modifications. Native MS 29,30 reveals that the interface between the C-terminal region and the ACD is dramatically weakened upon conversion of L-to D-Ser.…”

Section: Introductionmentioning

confidence: 99%

Structural and Functional Consequences of Age-Related Isomerization in α-Crystallins

Lyon

Riggs

Collier

et al. 2018

Preprint

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Long-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as isomerization. Recently, tandem-mass spectrometry approaches have enabled the identification and detailed characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here we examine the site-specific impact of isomerization of aspartic acid and epimerization of serine in human αAand αB-crystallin.From a total of 81 sites of modification identified in aged eye lenses, four ( αB Ser59, αA Ser162, αB Asp62, αB Asp109) map to crucial oligomeric interfaces. To characterize the effect of isomerization on quaternary assembly, molecular dynamics calculations and native mass spectrometry experiments were performed on recombinant forms of αAand αB-crystallin that incorporate, or mimic, isomerized residues. In all cases, oligomerization is significantly affected, with epimerization of a single serine residue ( αA Ser162) sufficing to weaken inter-subunit binding dramatically. Furthermore, phosphorylation of αB Ser59, known to play an important regulatory role in oligomerization, is severely inhibited by serine epimerization and altered by isomerization of nearby αB Asp62. Similarly, isomerization of αB Asp109 disrupts a vital salt-bridge with αB Arg120, a loss previously shown to yield aberrant oligomerization and aggregation in several disease variants. Our results illustrate how isomerization of amino-acid residues, which may seem like a minor structural perturbation, can have profound consequences on protein assembly and activity by disrupting specific hydrogen bonds and salt bridges. Significance StatementProteins play numerous critical roles in our bodies but suffer damage with increasing age. For example, isomerization is a spontaneous post-translational modification that alters the threedimensional connectivity of an amino acid, yet remains invisible to traditional proteomic experiments.Herein, radical-based fragmentation was used for isomer identification while molecular dynamics and native mass spectrometry were utilized to assess structural consequences. The results demonstrate that isomerization disrupts both oligomeric assembly and phosphorylation in the α-crystallins, which are long-lived proteins in the lens of the eye. The loss of function associated with these modifications is likely connected to age-related diseases such as cataract and neurodegenerative disorders, while the methodologies we present represent a framework for structure-function studies on other isomerized proteins.

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Mass spectrometry beyond the native state

Cited by 48 publications

References 79 publications

Processing of the SARS-CoV pp1a/ab nsp7–10 region

Processing of the SARS-CoV pp1a/ab nsp7–10 region

Phosphorylation of HspB1 regulates its mechanosensitive molecular chaperone interaction with native filamin C

Structural and Functional Consequences of Age-Related Isomerization in α-Crystallins

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