@article{Lanzicher2020Singl-49714,
  year={2020},
  doi={10.3389/fphys.2020.00173},
  title={Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP},
  volume={11},
  journal={Frontiers in Physiology},
  author={Lanzicher, Thomas and Zhou, Tiankun and Saripalli, Chandra and Keschrumrus, Vic and Smith, John E. and Mayans, Olga and Sbaizero, Orfeo and Granzier, Henk},
  note={Article Number: 173}
}

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    <dc:creator>Smith, John E.</dc:creator>
    <dcterms:title>Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP</dcterms:title>
    <dc:creator>Lanzicher, Thomas</dc:creator>
    <dc:contributor>Smith, John E.</dc:contributor>
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    <dc:contributor>Sbaizero, Orfeo</dc:contributor>
    <dcterms:issued>2020</dcterms:issued>
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    <dc:contributor>Zhou, Tiankun</dc:contributor>
    <dc:contributor>Lanzicher, Thomas</dc:contributor>
    <dc:creator>Mayans, Olga</dc:creator>
    <dcterms:rights rdf:resource="http://creativecommons.org/licenses/by/4.0/"/>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2020-05-28T08:56:17Z</dc:date>
    <dc:creator>Zhou, Tiankun</dc:creator>
    <dcterms:abstract xml:lang="eng">Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (F&lt;sub&gt;unfold&lt;/sub&gt;). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling.</dcterms:abstract>
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    <dc:creator>Saripalli, Chandra</dc:creator>
    <dc:contributor>Mayans, Olga</dc:contributor>
    <dc:contributor>Saripalli, Chandra</dc:contributor>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2020-05-28T08:56:17Z</dcterms:available>
    <dc:contributor>Keschrumrus, Vic</dc:contributor>
    <dc:creator>Granzier, Henk</dc:creator>
    <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/49714"/>
    <dc:creator>Keschrumrus, Vic</dc:creator>
    <dc:creator>Sbaizero, Orfeo</dc:creator>
    <dc:contributor>Granzier, Henk</dc:contributor>
    <dc:language>eng</dc:language>
    <dc:rights>Attribution 4.0 International</dc:rights>
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