Phase separation of a microtubule plus-end tracking protein into a fluid fractal network

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Jan. 30, 2025

Abstract Microtubule plus-end tracking proteins (+TIPs) participate in nearly all microtubule-based cellular processes and have recently been proposed to function as liquid condensates. However, their formation internal organization remain poorly understood. Here, we study the phase separation of Bik1, a CLIP-170 family member key +TIP involved budding yeast cell division. Bik1 is dimer with rod-shaped conformation primarily defined by its central coiled-coil domain. Its condensation likely involves higher-order oligomers that separate manner dependent on protein’s N-terminal CAP-Gly domain C-terminal EEY/F-like motif. This process accompanied conformational rearrangements leading at least two-fold increase multivalent interactions between folded disordered domains. Unlike classical liquids, condensates exhibit heterogeneous, fractal supramolecular structure protein- solvent-rich regions. structural evidence supports recent percolation-based models biomolecular Together, our findings offer insights into structure, dynamic rearrangement, complex, oligomeric, multidomain protein both dilute condensed states. Our experimental framework can be applied other condensates, including more complex networks.

Language: Английский

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Rational Modification of a Cross-Linker for Improved Flexible Protein Structure Modeling

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 9, 2025

Chemical cross-linking/mass spectrometry (XL-MS) has emerged as a complementary tool for mapping interaction sites within protein networks well gaining moderate-resolution native structural insight with minimal interference. XL-MS technology mostly relies on chemoselective reactions (cross-linking) between residues and linker. DSSO represents versatile cross-linker structure investigation in-cell XL-MS. However, our assessment of its shelf life batch purity revealed decomposition in anhydrous solution via retro-Michael reaction, which may reduce the active ingredient down to below 90%. To mitigate occurrence this degradative mechanism, we report rational design synthesis DSSO-carbamate, contains an inserted nitrogen atom backbone structure. This modification yielded remarkably favorable stability against such decomposition, translated higher cross-link monolink recovery when performing monomeric flexible proteins. Recently, been leveraged AlphaFold2 other prediction algorithms improved multiconformational end, demonstrate that novel cross-linker, termed generated more accurate predictions combined AlphaFold2, account increased cross-links monolinks, compared DSSO. As such, DSSO-carbamate useful addition community, particularly prediction.

Language: Английский

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Emerging Approaches to Investigating Functional Protein Dynamics in Modular Redox Enzymes: Nitric Oxide Synthase as a Model System

Journal of Biological Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 108282 - 108282

Published: Feb. 1, 2025

Language: Английский

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Mass Spectrometry‐Based Proteomics Technologies to Define Endogenous Protein‐Protein Interactions and Their Applications to Cancer and Viral Infectious Diseases

Mass Spectrometry Reviews, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 9, 2025

An intricate network of protein assemblies and protein-protein interactions (PPIs) underlies nearly every biological process in living systems. The organization these cellular networks is highly dynamic intimately tied to the genomic proteomic landscapes a cell. Disruptions normal PPIs can impair functions contribute development human diseases. In recent years, targeting has emerged as an attractive strategy for drug discovery. Consequently, identification characterization endogenous PPIs-those occurring naturally under physiological conditions-has become crucial unraveling molecular mechanisms driving pathology laying groundwork novel diagnostics therapeutics. Owing numerous technological advancements, mass spectrometry (MS)-based proteomics transformed study at systems-level. This review focuses on approaches that enable physiologically relevant interactions, spanning complex-centric structure-centric analyses. Additionally, their applications define native contexts cancer viral infectious diseases highlighted.

Language: Английский

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Cross‐Linking Mass Spectrometry of the Antimicrobial Peptides Magainin 2 and PGLa Reveals Heterodimerization in Micellar Medium

Rapid Communications in Mass Spectrometry, Journal Year: 2025, Volume and Issue: unknown

Published: April 27, 2025

ABSTRACT Rationale In this study, we applied cross‐linking mass spectrometry (XL‐MS) to characterize the oligomeric states of a PGLa/magainin 2 mixture and gain insight into heterodimerization previously suggested in literature. Both peptides have shown synergistic enhancement activity when tested antimicrobial assays; however, mechanism action is still not well understood. Methods Peptides solutions were prepared HEPES buffer presence membrane‐mimicking DDM detergent micelles or POPE:POPG 3:1 vesicles. Cross‐linking experiments performed using disuccinimidyl suberate (DSS) glutarate (DSG), MALDI‐MS was used follow performance. Nano liquid chromatography coupled conducted on Q Exactive Plus orbitrap achieve linkage sites determination pLink2 for data interpretation. Trypsin pepsin digestion characterization intermolecular links. Results XL‐MS micelle environment provided direct evidence specific heterodimer, but no other detected. Monitoring reaction allowed unambiguous cross‐linked stabilized oligomers facilitated rapid optimization conditions best balance between stabilizing complex formation avoiding unspecific aggregation. Comparison species lipidic bilayers revealed different behaviors suggesting that interaction might occur differently both media. Conclusions This study relevant at peptidomic level. However, workflow had be adjusted compared its use large‐scale protein–protein mapping order avoid technical bias arising from nature reaction.

Language: Английский

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Mass Spectrometry Structural Proteomics Enabled by Limited Proteolysis and Cross‐Linking

Mass Spectrometry Reviews, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 19, 2024

ABSTRACT The exploration of protein structure and function stands at the forefront life science represents an ever‐expanding focus in development proteomics. As mass spectrometry (MS) offers readout conformational changes both peptide levels, MS‐based structural proteomics is making significant strides realms molecular biology, complementing traditional biology techniques. This review focuses on two powerful techniques for peptide‐level readout, namely limited proteolysis‐mass (LiP‐MS) cross‐linking (XL‐MS). First, we discuss principles, features, different workflows these methods. Subsequently, delve into bioinformatics strategies software tools used interpreting data associated with conformation readouts how can be integrated other computational tools. Furthermore, provide a comprehensive summary noteworthy applications LiP‐MS XL‐MS diverse areas including neurodegenerative diseases, interactome studies, membrane proteins, artificial intelligence‐based analysis. Finally, factors that modulate changes. We also highlight remaining challenges understanding intricacies by technologies.

Language: Английский

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Autorepression of yeast Hsp70 cochaperones by intramolecular interactions involving their J-domains

Cell Stress and Chaperones, Journal Year: 2024, Volume and Issue: 29(2), P. 338 - 348

Published: March 21, 2024

The Hsp70 chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) co-evolved with Hsp70s to trigger ATP-hydrolysis and catalytically upload various substrate polypeptides need be structurally modified by the chaperone. Here, we measured disaggregation refolding activities of main yeast cytosolic Hsp70, Ssa1, presence its most abundant JDPs, Sis1 Ydj1, two swap mutants, which J-domains have been interchanged. observed differences four constructs differently cooperate Ssa1 each other, as well their intrinsic ability bind misfolded substrates Ssa1's ATPase, indicate yet uncharacterized intra-molecular dynamic interactions between remaining C-terminal segments these proteins. Taken together, data suggest an auto-regulatory role within both type A B might evolved reduce energy-costly ATPase cycles Ssa1-4 that are cytosol.

Language: Английский

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δ‐Conotoxin Structure Prediction and Analysis through Large‐Scale Comparative and Deep Learning Modeling Approaches

Advanced Science, Journal Year: 2024, Volume and Issue: unknown

Published: July 21, 2024

The δ-conotoxins, a class of peptides produced in the venom cone snails, are interest due to their ability inhibit inactivation voltage-gated sodium channels causing paralysis and other neurological responses, but difficulties isolation synthesis have made structural characterization challenging. Taking advantage recent breakthroughs computational algorithms for structure prediction that modeling especially useful when experimental data is sparse, this work uses both deep-learning-based algorithm AlphaFold comparative method RosettaCM model analyze 18 previously uncharacterized δ-conotoxins derived from piscivorous, vermivorous, molluscivorous snails. models provide insights into aspects these suggest features likely be significant influencing binding different pharmacological activities against targets, with implications drug development. Additionally, described protocol provides roadmap similar disulfide-rich by complementary methods.

Language: Английский

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Recent Advances in Mass Spectrometry-based Protein Interactome Studies

Molecular & Cellular Proteomics, Journal Year: 2024, Volume and Issue: unknown, P. 100887 - 100887

Published: Nov. 1, 2024

Language: Английский

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Phase separation of a microtubule plus-end tracking protein into a fluid fractal network

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 19, 2024

Abstract Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based cellular processes, and it has been recently proposed that they function as liquid condensates. However, the formation process internal organization of +TIP condensates poorly understood. Here, we have investigated phase separation CLIP-170 family member Bik1, a key implicated budding yeast cell division. We found Bik1 is rod-shaped dimer whose conformation dominated by its central coiled-coil domain. Liquid condensation accompanied conformational rearrangements, leading to 2-3-fold rise interactions between protein’s folded disordered domains. In contrast classical liquids, supramolecular structure condensate heterogeneous, with fractal protein-rich protein-free This observation provides structural evidence support recent models biomolecular based on percolation. More broadly, our results provide insights into structure, dynamic rearrangement, complex, multidomain protein dilute condensed phases. Our experimental framework can be extended other condensates, including more intricate networks.

Language: Английский

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