Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP
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) flank...
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Frontiers Media S.A.
2020-03-01
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| Series: | Frontiers in Physiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fphys.2020.00173/full |
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| author | Thomas Lanzicher Thomas Lanzicher Tiankun Zhou Chandra Saripalli Vic Keschrumrus John E. Smith III Olga Mayans Orfeo Sbaizero Henk Granzier |
| author_facet | Thomas Lanzicher Thomas Lanzicher Tiankun Zhou Chandra Saripalli Vic Keschrumrus John E. Smith III Olga Mayans Orfeo Sbaizero Henk Granzier |
| author_sort | Thomas Lanzicher |
| collection | DOAJ |
| description | 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 (Funfold). 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. |
| first_indexed | 2024-12-11T11:59:36Z |
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| id | doaj.art-4b14674ac8e34676a1fa21d9d3f71407 |
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| issn | 1664-042X |
| language | English |
| last_indexed | 2024-12-11T11:59:36Z |
| publishDate | 2020-03-01 |
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| spelling | doaj.art-4b14674ac8e34676a1fa21d9d3f714072022-12-22T01:08:07ZengFrontiers Media S.A.Frontiers in Physiology1664-042X2020-03-011110.3389/fphys.2020.00173522261Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARPThomas Lanzicher0Thomas Lanzicher1Tiankun Zhou2Chandra Saripalli3Vic Keschrumrus4John E. Smith III5Olga Mayans6Orfeo Sbaizero7Henk Granzier8Department of Cellular & Molecular Medicine, The University of Arizona, Tucson, AZ, United StatesDepartment of Engineering and Architecture, University of Trieste, Trieste, ItalyDepartment of Biology, University of Konstanz, Konstanz, GermanyDepartment of Cellular & Molecular Medicine, The University of Arizona, Tucson, AZ, United StatesDepartment of Cellular & Molecular Medicine, The University of Arizona, Tucson, AZ, United StatesDepartment of Cellular & Molecular Medicine, The University of Arizona, Tucson, AZ, United StatesDepartment of Biology, University of Konstanz, Konstanz, GermanyDepartment of Engineering and Architecture, University of Trieste, Trieste, ItalyDepartment of Cellular & Molecular Medicine, The University of Arizona, Tucson, AZ, United StatesTitin 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 (Funfold). 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.https://www.frontiersin.org/article/10.3389/fphys.2020.00173/fulltitinpassive stiffnessspring elementspost-translational modificationmechano-signaling |
| spellingShingle | Thomas Lanzicher Thomas Lanzicher Tiankun Zhou Chandra Saripalli Vic Keschrumrus John E. Smith III Olga Mayans Orfeo Sbaizero Henk Granzier Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP Frontiers in Physiology titin passive stiffness spring elements post-translational modification mechano-signaling |
| title | Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP |
| title_full | Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP |
| title_fullStr | Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP |
| title_full_unstemmed | Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP |
| title_short | Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP |
| title_sort | single molecule force spectroscopy on the n2a element of titin effects of phosphorylation and carp |
| topic | titin passive stiffness spring elements post-translational modification mechano-signaling |
| url | https://www.frontiersin.org/article/10.3389/fphys.2020.00173/full |
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