Extensive and Modular Intrinsically Disordered Segments in C. elegans TTN-1 and Implications in Filament Binding, Elasticity and Oblique Striation

• Published in: Journal of molecular biology 2010-05, Vol.398 (5), p.672-689
• Main Authors: Forbes, Jeffrey G., Flaherty, Denise B., Ma, Kan, Qadota, Hiroshi, Benian, Guy M., Wang, Kuan
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies - immunology; C. elegans titin; Caenorhabditis elegans - chemistry; Caenorhabditis elegans - physiology; Circular Dichroism; Connectin; Elasticity; force sensor; Microscopy, Fluorescence; Models, Biological; Models, Molecular; Molecular Sequence Data; Muscle Proteins - chemistry; Muscle Proteins - metabolism; polyproline II helix; Protein Binding; Protein Conformation; Protein Kinases - chemistry; Protein Kinases - metabolism; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; staggered helical bundle; Staining and Labeling - methods; Temperature
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2010.03.032

TTN-1, a titin like protein in Caenorhabditis elegans, is encoded by a single gene and consists of multiple Ig and fibronectin 3 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 an ∼ 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 display a combination of a polyproline II helix and an unordered structure in aqueous solution and convert in trifluoroethanol to α-helix (PEVT, CEEEI, DispRep) and β-turn (AAPLE) structures, respectively. The octads in BLUE motifs form unstable α-helix-like structures coils in aqueous solution and negligible heptad-based, α-helical coiled-coils. 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, CEEEI and DispRep regions are all intrinsically disordered and highly reminiscent of the conformational malleability and elasticity of vertebrate titin PEVK segments. The proposed presence of long, modular and unstable α-helical oligomerization domains in the BLUE region of TTN-1 could bundle TTN-1 and stabilize oblique striation of the sarcomere.