Extensive and modular intrinsically disordered segments in C. elegans TTN-1 and implications in filament binding, elasticity and oblique striation

• Type of Material: Article
• Authors: Jeffrey G. Forbes, National Institutes of Health; Denise B. Flaherty, Emory University; Kan Ma, National Institutes of Health; Hiroshi Kadota, Emory University; Guy Benian, Emory University; Kuan Wang, National Institutes of Health
• Language: English
• Date: 2010-05-21
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2010, Elsevier
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1016/j.jmb.2010.03.032
• Title of Journal or Parent Work: Journal of Molecular Biology
• ISSN: 0022-2836
• Volume: 398
• Issue: 5
• Start Page: 672
• End Page: 689
• Grant/Funding Information: This work was supported in part to KW by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, DHHS and to GB by NIAMS/NIH grant AR051466.
• Keywords: polyproline II helix; force sensor; C. elegans titin; Circular dichroism; passive tension; staggered helical bundle; intrinsically disordered proteins
• Research Categories: Health Sciences, Pathology; Biology, Molecular

Abstract

TTN-1, a predicted titin-like protein in C. elegans, is encoded by a single gene, and consists of multiple Ig and Fn3 domains, a protein kinase domain and several regions containing tandem short repeat sequences. We have characterized TTN-1’s sarcomere distribution, protein interaction with key myofibrillar proteins as well as the conformation malleability of representative motifs of five classes of short repeats. We report that two antibodies developed to portions of TTN-1 detect a ~2 MDa polypeptide on western blots. In addition, by immunofluorescence staining, both of these antibodies localize to the I-band and may extend into the outer edge of the A-band in the obliquely striated muscle of the nematode. Six different 300 residue segments of TTN-1 were shown to variously interact with actin and/or myosin in vitro. Conformations of synthetic peptides of representative copies of each of the five classes of repeats: 39-mer PEVT, 51-mer CEEEI, 42-mer AAPLE, 32-mer BLUE and 30-mer DispRep were investigated by circular dichroism at different temperatures, ionic strengths and solvent polarities. The PEVT, CEEEI, DispRep and AAPLE peptides displayed a combination of a polyproline II helix and an unordered structure in aqueous solution and converts in trifluoroethanol to α-helix (PEVT, DispRep) and β-turn (AAPLE) structures, respectively. The octads in BLUE motifs form unstable α-helix-unstructured coil in aqueous solution and negligible heptad-based coiled-coils, as predicted based on sequence analysis. The α-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface. Our finding indicates that APPLE, PEVT, DispRep regions are all intrinsically disordered and highly reminiscent of the conformational malleability and elasticity of vertebrate titin PEVK segments. The presence of long, modular and unstable α-helical oligomerization domains in the BLUE region of TTN-1 could oligomerize and bundle TTN-1 and stabilize oblique striation of the sarcomere.