Apolipoprotein E4 has extensive conformational heterogeneity in lipid free and bound forms

Stuchell‐Brereton, Melissa D.; Zimmerman, Maxwell I.; Miller, Jenna; Mallimadugula, Upasana L.; Incicco, Jeremias; Roy, Debjit; Smith, Louis G.; Baban, Berevan; DeKoster, Gregory T.; Frieden, Carl; Bowman, Gregory R.; Soranno, Andrea

doi:10.1101/2022.02.02.478828

Cited by 2 publications

(6 citation statements)

References 68 publications

(96 reference statements)

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“…Single-molecule FRET measurements were performed on both a modified Picoquant MT200 instrument (Picoquant, Berlin, Germany) and an equivalent custom-built instrument. Specifications of the instrumentation were previously published (34), and we briefly summarize them here.…”

Section: Methodsmentioning

confidence: 99%

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

Cubuk,

Greenberg,

Greenberg

et al. 2024

Preprint

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

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Section: Methodsmentioning

confidence: 99%

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

Cubuk,

Greenberg,

Greenberg

et al. 2024

Preprint

Self Cite

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“…Single-molecule FRET measurements were performed on a modified Picoquant MT200 instrument (Picoquant, Berlin, Germany) as described 15 . Experiments were conducted at a protein concentration of 100-250 pM (estimated from dilutions of samples with known concentration based on absorbance (which was not certified by peer review) is the author/funder.…”

Section: Single-molecule Fret Experimentsmentioning

confidence: 99%

“…Single-molecule FRET data were analyzed using the "Fretica" package developed by Daniel Nettels and Ben Schuler (University of Zurich, Zurich, CH) and available online at https://schuler.bioc.uzh.ch/wpcontent/uploads/2020/09/Fretica20200915.zip. Fluorescence lifetimes were obtained via a convolution with the Instrument Response Function (IRF), measured as described 15 . Time resolved anisotropies were computed as described 15 .…”

Section: Single-molecule Fret Experimentsmentioning

confidence: 99%

“…Fluorescence lifetimes were obtained via a convolution with the Instrument Response Function (IRF), measured as described 15 . Time resolved anisotropies were computed as described 15 . Fits for titration curves and 95% confidence intervals were calculated using Mathematica (Princeton, NJ).…”

Section: Single-molecule Fret Experimentsmentioning

confidence: 99%

“…Based on these considerations, we identified the two sets of labeling positions: CP α1N44C β2E84C ("α44β84") and CP α1S9C β2S161C ("α9β161") (Figure 1). We labeled the CP samples with a sequential labeling procedure 15,33 . Substoichiometric dye concentrations were used for donor dye coupling, followed by purification of single-donor-labeled CP, and then coupling acceptor dye with a molar excess of dye.…”

Section: Design Of Labeling Positions On Cpmentioning

confidence: 99%

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Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

Mooren,

Stuchell-Brereton,

McConnell

et al. 2023

Preprint

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Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Forster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked- binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

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Apolipoprotein E4 has extensive conformational heterogeneity in lipid free and bound forms

Cited by 2 publications

References 68 publications

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

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

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

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