scholarly journals Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

2020 ◽  
Author(s):  
Nuwani W. Weerasinghe ◽  
Yeganeh Habibi ◽  
Kevin A. Uggowitzer ◽  
Christopher J. Thibodeaux
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Conformational Changes ◽  
Ion Mobility ◽  
Peptide Binding ◽  
Native Mass Spectrometry ◽  
Order Structure ◽  
Conformational Sampling ◽  
Precursor Peptide ◽  
Conformational Landscape

AbstractLanthipeptides are ribosomally-synthesized and post-translationally modified peptide (RiPP) natural products that are biosynthesized in a multistep maturation process by enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding, and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These “biophysical” processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native nanoelectrospray ionization coupled to ion mobility mass spectrometry (nanoESI-IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its non-covalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively-folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme, and that the precursor peptide itself exhibits higher order structure in the mass spectrometer. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes nanoESI-IM-MS as a versatile approach for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.

scholarly journals State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

2021 ◽  
Vol 14 (6) ◽  
pp. 498
Author(s):  
Evolène Deslignière ◽  
Anthony Ehkirch ◽  
Bastiaan L. Duivelshof ◽  
Hanna Toftevall ◽  
Jonathan Sjögren ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility ◽  
State Of The Art ◽  
Native Mass Spectrometry ◽  
Site Specific ◽  
Drug Conjugates ◽  
Conjugation Process ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

scholarly journals Extending native mass spectrometry approaches to integral membrane proteins

2015 ◽  
Vol 396 (9-10) ◽  
pp. 991-1002 ◽  
Author(s):  
Albert Konijnenberg ◽  
Jeroen F. van Dyck ◽  
Lyn L. Kailing ◽  
Frank Sobott
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Gas Phase ◽  
Conformational Changes ◽  
Drug Targets ◽  
Native Mass Spectrometry ◽  
Lipid Binding ◽  
Drug Binding ◽  
Integral Membrane Proteins ◽  
Oligomeric State

Abstract Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergent-based reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins.

scholarly journals DNA and RNA Telomeric G-Quadruplexes: What Topology Features Can be Inferred from Ion Mobility Mass Spectrometry?

2019 ◽  
Author(s):  
Valentina D’Atri ◽  
Valerie Gabelica
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility Spectrometry ◽  
Ion Mobility ◽  
Native Mass Spectrometry ◽  
Cation Binding ◽  
Telomeric Dna ◽  
Dna And Rna ◽  
G Quadruplex ◽  
Multiple Structures

Maintenance of the telomeres is key to chromosome integrity and cell proliferation. The G-quadruplex structures formed by telomeric DNA and RNA (TTAGGG and UUAGGG repeats, respectively) are key to this process. However, because these sequences are particularly polymorphic, solving high-resolution structures is not always possible, and there is a need for new methodologies to characterize the multiple structures coexisting in solution. In this context, we evaluated whether ion mobility spectrometry coupled to native mass spectrometry could help separate and assign the G-quadruplex topologies. We explored the circular dichroism spectra, multimer formation, cation binding, and ion mobility spectra of several 4-repeat and 8-repeat telomeric DNA and RNA sequences, both in NH<sub>4</sub><sup>+</sup> and in K<sup>+</sup>. In 1 mM K<sup>+</sup> and 100 mM trimethylammonium acetate, all RNAs fold intramolecularly (no multimer). In 8-repeat sequences, the subunits are not independent: in DNA the first subunit disfavors the folding of the second one, whereas in RNA the two subunits fold cooperatively via cation-mediated stacking. Ion mobility spectrometry shows that gas-phase structures keep a memory of the solution ones, but not identical. At the native charge states, the loops can rearrange in a variety of ways (unless they are constrained by pre-formed hydrogen bonds), thereby wrapping the core and masking the strand arrangements. Our study highlights that, to progress towards structural assignment from IM-MS experiments, deeper understanding of the solution-to-gas-phase rearrangement mechanisms is warranted. <br>

scholarly journals DNA and RNA Telomeric G-Quadruplexes: What Topology Features Can be Inferred from Ion Mobility Mass Spectrometry?

2019 ◽  
Author(s):  
Valentina D’Atri ◽  
Valerie Gabelica
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility Spectrometry ◽  
Ion Mobility ◽  
Native Mass Spectrometry ◽  
Cation Binding ◽  
Telomeric Dna ◽  
Dna And Rna ◽  
G Quadruplex ◽  
Multiple Structures

Maintenance of the telomeres is key to chromosome integrity and cell proliferation. The G-quadruplex structures formed by telomeric DNA and RNA (TTAGGG and UUAGGG repeats, respectively) are key to this process. However, because these sequences are particularly polymorphic, solving high-resolution structures is not always possible, and there is a need for new methodologies to characterize the multiple structures coexisting in solution. In this context, we evaluated whether ion mobility spectrometry coupled to native mass spectrometry could help separate and assign the G-quadruplex topologies. We explored the circular dichroism spectra, multimer formation, cation binding, and ion mobility spectra of several 4-repeat and 8-repeat telomeric DNA and RNA sequences, both in NH<sub>4</sub><sup>+</sup> and in K<sup>+</sup>. In 1 mM K<sup>+</sup> and 100 mM trimethylammonium acetate, all RNAs fold intramolecularly (no multimer). In 8-repeat sequences, the subunits are not independent: in DNA the first subunit disfavors the folding of the second one, whereas in RNA the two subunits fold cooperatively via cation-mediated stacking. Ion mobility spectrometry shows that gas-phase structures keep a memory of the solution ones, but not identical. At the native charge states, the loops can rearrange in a variety of ways (unless they are constrained by pre-formed hydrogen bonds), thereby wrapping the core and masking the strand arrangements. Our study highlights that, to progress towards structural assignment from IM-MS experiments, deeper understanding of the solution-to-gas-phase rearrangement mechanisms is warranted. <br>

scholarly journals Ion mobility coupled to native mass spectrometry as a relevant tool to investigate extremely small ligand-induced conformational changes

The Analyst ◽  
2015 ◽  
Vol 140 (21) ◽  
pp. 7234-7245 ◽  
Author(s):  
Johann Stojko ◽  
Sonia Fieulaine ◽  
Stéphanie Petiot-Bécard ◽  
Alain Van Dorsselaer ◽  
Thierry Meinnel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Ion Mobility ◽  
Native Mass Spectrometry ◽  
Peptide Deformylase ◽  
Ion Mobility Mass Spectrometry ◽  
Over Time

Native and ion-mobility mass spectrometry reveal the conformational evolution over time of a peptide deformylase binding different ligands, which is consistent with slow-tight inhibition of the enzyme.

scholarly journals Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility–mass spectrometry

2019 ◽  
Vol 476 (21) ◽  
pp. 3125-3139 ◽  
Author(s):  
Daniel Shiu-Hin Chan ◽  
Jeannine Hess ◽  
Elen Shaw ◽  
Christina Spry ◽  
Robert Starley ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Structural Biology ◽  
Ion Mobility ◽  
Biosynthetic Pathway ◽  
Screening Tool ◽  
Native Mass Spectrometry ◽  
Ion Mobility Mass Spectrometry ◽  
E Coli ◽  
Structural Insights

Abstract CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility–mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

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