Structure Characterization of a Disordered Peptide Using In-Droplet Hydrogen/Deuterium Exchange Mass Spectrometry and Molecular Dynamics

ACS Physical Chemistry Au, Journal Year: 2024, Volume and Issue: 5(1), P. 17 - 29

Published: Nov. 13, 2024

In-droplet hydrogen/deuterium exchange (HDX)-mass spectrometry (MS) experiments have been conducted for peptides of highly varied conformational type. A new model is presented that combines the use protection factors (PF) from molecular dynamics (MD) simulations with intrinsic HDX rates (kint) to obtain a structure-to-reactivity calibration curve. Using model, relationship peptide structural flexibility and reactivity different elucidated. Additionally, used describe degree bias disease-relevant Nt17 peptide; although flexible, intrinsically primed facile conversion α-helical conformation upon binding partners imparts significant in-droplet this peptide. In future, scale may be developed whereby predictive (propensity form 2° elements such as α-helix, β-sheet, β-turn) disordered regions (IDRs). Such resolution ultimately high-throughput screening IDR transformation(s) ligands drug candidates.

Language: Английский

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Identifying Sequence Effects on Chain Dimensions of Disordered Proteins by Integrating Experiments and Simulations

JACS Au, Journal Year: 2024, Volume and Issue: 4(12), P. 4729 - 4743

Published: Nov. 14, 2024

It has become increasingly evident that the conformational distributions of intrinsically disordered proteins or regions are strongly dependent on their amino acid compositions and sequence. To facilitate a systematic investigation these sequence-ensemble relationships, we selected set 16 naturally occurring identical length but with large differences in composition, hydrophobicity, charge patterning. We probed ensembles single-molecule Förster resonance energy transfer (FRET), complemented by circular dichroism (CD) nuclear magnetic (NMR) spectroscopy as well small-angle X-ray scattering (SAXS). The shows strong dependence chain dimensions sequence volumes differing up to factor 6. residue-specific intrachain interaction networks underlie pronounced were identified using atomistic simulations combined ensemble reweighting, revealing important role charged, aromatic, polar residues. advance transferable description protein regions, further employed experimental data parametrize coarse-grained model for includes an explicit representation FRET fluorophores successfully describes experiments different dye pairs. Our findings demonstrate value integrating advancing our quantitative understanding features determine proteins.

Language: Английский

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Structural dynamics of the intrinsically disordered linker region of cardiac troponin T

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

Published: May 31, 2024

The cardiac troponin complex, composed of troponins I, T, and C, plays a central role in regulating the calcium-dependent interactions between myosin thin filament. Mutations can cause cardiomyopathies; however, it is still major challenge to connect how changes sequence affect troponin's function. Recent high-resolution structures filament revealed critical insights into structure-function relationship troponin, but there remain large, unresolved segments including troponin-T linker region that hotspot for cardiomyopathy mutations. This predicted be intrinsically disordered, with behaviors are not well described by traditional structural approaches; this proposal has been experimentally verified. Here, we used combination single-molecule Förster resonance energy transfer (FRET), molecular dynamics simulations, functional reconstitution assays investigate region. We show context both isolated fully regulated behaves as dynamic, disordered undergoes polyampholyte expansion presence high salt distinct conformational during assembly complex. also examine ΔE160 hypertrophic mutation demonstrate does linker, rather allosterically affects other complex subunits, leading increased contractility. Taken together, our data clearly importance disorder within provide new mechanisms driving pathogenesis cardiomyopathies.

Language: Английский

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Phosphorylation of disordered proteins tunes local and global intramolecular interactions

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

Published: June 12, 2024

ABSTRACT Protein post-translational modifications, such as phosphorylation, are important regulatory signals for diverse cellular functions. In particular, intrinsically disordered protein regions (IDRs) subject to phosphorylation a means modulate their interactions and Toward understanding the relationship between in IDRs specific functional outcomes, we must consider how affects IDR conformational ensemble. Various experimental techniques suited interrogate features of ensembles; molecular simulations can provide complementary insights even illuminate ensemble that may be experimentally inaccessible. Therefore, sought expand tools available study phosphorylated by all-atom Monte Carlo simulations. To this end, implemented parameters phosphoserine (pSer) phosphothreonine (pThr) into OPLS version continuum solvent model, ABSINTH, assessed performance compared published findings. We simulated short (< 20 residues) long (> 80 phospho-IDRs that, collectively, survey both local global phosphorylation-induced changes Our four well-studied show near-quantitative agreement with findings these systems via metrics including radius gyration, transient helicity, persistence length. also leveraged inherent advantage sequence control explore effects combinations phospho-sites two multi-phosphorylated IDRs. results support on prior observations connect Herein, describe alter chemistry, net charge patterning, intramolecular interactions, which collectively features. SIGNIFICANCE Spatially temporally controlled is critical many facets function broader health. Intrinsically overrepresented targets but structural consequences modifications remain elusive systems. rigorous modeling using simulations, present new ABSINTH implicit paradigm. Through example phospho-IDRs, demonstrate excellent our phospho-IDR datasets.

Language: Английский

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The dimerization domain of SARS CoV 2 Nucleocapsid protein is partially disordered as a monomer and forms a high affinity dynamic complex.

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

Published: Sept. 25, 2024

Abstract The SARS-CoV-2 Nucleocapsid (N) is a 419 amino acids protein that drives the compaction and packaging of viral genome. This aided not only by protein-RNA interactions, but also protein-protein interactions contribute to increasing valence nucleocapsid protein. Here, we focused on quantifying mechanisms control dimer formation. Single-molecule Förster Resonance Energy Transfer enabled us investigate conformations dimerization domain in context full-length as well energetics associated with dimerization. Under monomeric conditions, observed significantly expanded configurations (compared folded structure), which are consistent dynamic conformational ensemble. addition unlabeled stabilizes configuration high mean transfer efficiency, agreement predictions based known structures. Dimerization characterized dissociation constant ∼ 12 nM at 23 O C driven strong enthalpic between two subunits, originate from coupled folding binding. Interestingly, structure retains some heterogeneity units, denaturant reveals can expand before being completely destabilized. Our findings suggest inherent flexibility monomer form required adopt specific fold domain, where subunits interlock one another. We proposed retained may favor capture RNA, temperature dependence explain previous observations regarding phase separation propensity N

Language: Английский

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