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Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP

Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP

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LANZICHER, Thomas, Tiankun ZHOU, Chandra SARIPALLI, Vic KESCHRUMRUS, John E. SMITH, Olga MAYANS, Orfeo SBAIZERO, Henk GRANZIER, 2020. Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP. In: Frontiers in Physiology. Frontiers Research Foundation. 11, 173. eISSN 1664-042X. Available under: doi: 10.3389/fphys.2020.00173

@article{Lanzicher2020Singl-49714, title={Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP}, year={2020}, doi={10.3389/fphys.2020.00173}, 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} }

Lanzicher, Thomas Lanzicher, Thomas Granzier, Henk 2020 Smith, John E. Keschrumrus, Vic 2020-05-28T08:56:17Z Mayans, Olga Zhou, Tiankun Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP Attribution 4.0 International Mayans, Olga 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<sub>unfold</sub>). 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. 2020-05-28T08:56:17Z Keschrumrus, Vic Saripalli, Chandra Sbaizero, Orfeo Sbaizero, Orfeo Zhou, Tiankun eng Saripalli, Chandra Granzier, Henk Smith, John E.

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