scholarly journals Native Mass Spectrometry Reveals the Conformational Diversity of the UVR8 Photoreceptor

2018 ◽  
Author(s):  
Inês S. Camacho ◽  
Alina Theisen ◽  
Linus O. Johannissen ◽  
L. Aranzazú Díaz-Ramos ◽  
John M. Christie ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Uv Light ◽  
Native Mass Spectrometry ◽  
Full Length ◽  
Structural Investigations ◽  
Core Domain ◽  
Intrinsically Disordered ◽  
The Core ◽  
C Terminus ◽  
Conformational Diversity

AbstractUVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C-terminus is required for functional binding to signalling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photo-activation. We show that, whilst truncated UVR8 photo-converts from a single conformation of dimers to a single monomer conformation, the full-length protein exist in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C-terminus is primed for activation. In the monomer the extended C-terminus destabilises the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.TOC Graphic

scholarly journals Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor

2019 ◽  
Vol 116 (4) ◽  
pp. 1116-1125 ◽  
Author(s):  
Inês S. Camacho ◽  
Alina Theisen ◽  
Linus O. Johannissen ◽  
L. Aranzazú Díaz-Ramos ◽  
John M. Christie ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Uv Light ◽  
Native Mass Spectrometry ◽  
Full Length ◽  
Structural Investigations ◽  
Core Domain ◽  
Intrinsically Disordered ◽  
The Core ◽  
C Terminus ◽  
Conformational Diversity

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light, resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C terminus is required for functional binding to signaling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photoactivation. We show that, while truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exists in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C terminus is primed for activation. In the monomer the extended C terminus destabilizes the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.

scholarly journals Protein-based condensation mechanisms drive the assembly of RNA-rich P granules

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Helen Schmidt ◽  
Andrea Putnam ◽  
Dominique Rasoloson ◽  
Geraldine Seydoux
Keyword(s):  
Intrinsically Disordered Protein ◽  
P Granules ◽  
C Elegans ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
The Core ◽  
C Terminus ◽  
Germ Granules

Germ granules are protein-RNA condensates that segregate with the embryonic germline. In C. elegans embryos, germ (P) granule assembly requires MEG-3, an intrinsically-disordered protein that forms RNA-rich condensates on the surface of PGL condensates at the core of P granules. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). We find that MEG-3 is modular protein that uses its IDR to bind RNA and its C-terminus to drive condensation. The HMGL motif mediates binding to PGL-3 and is required for co-assembly of MEG-3 and PGL-3 condensates in vivo. Mutations in HMGL cause MEG-3 and PGL-3 to form separate condensates that no longer co-segregate to the germline or recruit RNA. Our findings highlight the importance of protein-based condensation mechanisms and condensate-condensate interactions in the assembly of RNA-rich germ granules.

scholarly journals Depicting Conformational Ensembles of α-Synuclein by Single Molecule Force Spectroscopy and Native Mass Spectroscopy

2019 ◽  
Vol 20 (20) ◽  
pp. 5181 ◽  
Author(s):  
Roberta Corti ◽  
Claudia A. Marrano ◽  
Domenico Salerno ◽  
Stefania Brocca ◽  
Antonino Natalello ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Single Molecule ◽  
Conformational Changes ◽  
Intrinsically Disordered Proteins ◽  
Force Spectroscopy ◽  
Native Mass Spectrometry ◽  
Disordered Proteins ◽  
Unique Contribution ◽  
Single Molecule Force Spectroscopy ◽  
Intrinsically Disordered

Description of heterogeneous molecular ensembles, such as intrinsically disordered proteins, represents a challenge in structural biology and an urgent question posed by biochemistry to interpret many physiologically important, regulatory mechanisms. Single-molecule techniques can provide a unique contribution to this field. This work applies single molecule force spectroscopy to probe conformational properties of α-synuclein in solution and its conformational changes induced by ligand binding. The goal is to compare data from such an approach with those obtained by native mass spectrometry. These two orthogonal, biophysical methods are found to deliver a complex picture, in which monomeric α-synuclein in solution spontaneously populates compact and partially compacted states, which are differently stabilized by binding to aggregation inhibitors, such as dopamine and epigallocatechin-3-gallate. Analyses by circular dichroism and Fourier-transform infrared spectroscopy show that these transitions do not involve formation of secondary structure. This comparative analysis provides support to structural interpretation of charge-state distributions obtained by native mass spectrometry and helps, in turn, defining the conformational components detected by single molecule force spectroscopy.

scholarly journals Zinc Binding to Tau Influences Aggregation Kinetics and Oligomer Distribution

2019 ◽  
Vol 20 (23) ◽  
pp. 5979 ◽  
Author(s):  
Moreira ◽  
Cristóvão ◽  
Torres ◽  
Carapeto ◽  
Rodrigues ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metal Ion ◽  
Molecular Mechanisms ◽  
Disease Onset ◽  
Native Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Zinc Binding ◽  
Lag Phase ◽  
Intrinsically Disordered ◽  
Tau Aggregation

Metal ions are well known modulators of protein aggregation and are key players in Alzheimer’s Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.

scholarly journals DNA binding reorganizes the intrinsically disordered C-terminal region of PSC in Drosophila PRC1

2020 ◽  
Author(s):  
Jin Joo Kang ◽  
Denis Faubert ◽  
Jonathan Boulais ◽  
Nicole J. Francis
Keyword(s):  
Mass Spectrometry ◽  
Dna Binding ◽  
Ubiquitin Ligase ◽  
Terminal Region ◽  
Cross Linking ◽  
Chromatin Binding ◽  
Protein Footprinting ◽  
Intrinsically Disordered ◽  
The Core ◽  
Binding Surface

AbstractPolycomb Group (PcG) proteins regulate gene expression by modifying chromatin. A key PcG complex, Polycomb Repressive Complex 1 (PRC1), has two activities: a ubiquitin ligase activity for histone H2A, and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal homology region (HR) of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase for H2A. The intrinsically disordered C-terminal region of PSC (PSC-CTR) compacts chromatin, and inhibits chromatin remodeling and transcription in vitro. Both the PSC-HR and the PSC-CTR are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used cross-linking mass spectrometry (XL-MS) to analyze the conformations of the PSC-CTR in PRC1 and how they change on binding DNA. XL-MS identifies interactions between the PSC-CTR and the core of PRC1, including between the PSC-CTR and PSC-HR. New contacts and overall more compacted PSC-CTR conformations are induced by DNA binding. Protein footprinting of accessible lysine residues in the PSC-CTR reveals an extended, bipartite candidate DNA/chromatin binding surface. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible PSC-CTR. Intramolecular interactions of the PSC-CTR detected by XL-MS can bring the high affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large IDRs that bind nucleic acids.HighlightsAn intrinsically disordered region (IDR) in Polycomb protein PSC compacts chromatinCross-linking mass spectrometry (XL-MS) was used to analyze topology of the PSC IDRProtein footprinting suggests a bipartite DNA binding surface in the PSC IDRA model for the DNA-driven organization of the PSC IDRCombining XL-MS and protein footprinting is a strategy to understand nucleic acid binding IDRsAbstract Figure

Pharmacokinetics of Full Length and Two-Domain Tissue Factor Pathway Inhibitor in Combination with Heparin in Rabbits

1993 ◽  
Vol 70 (03) ◽  
pp. 454-457 ◽  
Author(s):  
Claus Bregengaard ◽  
Ole Nordfang ◽  
Per Østergaard ◽  
Jens G L Petersen ◽  
Giorgio Meyn ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Fold Increase ◽  
Volume Of Distribution ◽  
Full Length ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
C Terminus ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus.FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg).Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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