ABSTRACT

Titin (connectin) is a giant protein that forms a single-molecule elastic filament extending from the M-line to the Z-line in the striated muscle sarcomere. The sequence of titin consists mainly of repeats of two types of 100-amino acid motifs (class I and class II that show homology to the fibronectin type III and immunoglobulin-C2 domains, respectively). To investigate the functions of the two classes of titin motifs as the basic units of this large sarcomere organizer molecule, titin cDNA segments encoding single class I or class II or linked class I-II motifs were cloned by polymerase chain reaction from a rat cardiac cDNA library into the T7 RNA polymerase-based pAED4 vector to express non-fusion titin fragments in Escherichia coli. High level expression of the three titin fragments was achieved, and effective rapid purification procedures were developed. The purified titin fragments were verified by their amino acid composition, apparent molecular mass, and charge. Antibodies raised against the genetically expressed titin motifs specifically recognized intact rat cardiac and skeletal muscle titins in Western blotting and immunofluorescence microscopy, confirming the authenticity of the cloned fragments. High -sheet contents of these titin motifs indicate a folding state very similar to that of intact native titin. Solid-phase protein-binding assays demonstrated that a single class I motif was able to bind both myosin and F-actin. In comparison, a single class II motif had weaker binding to only F-actin but the fragment containing linked class I and class II motifs showed significantly stronger interactions with both myosin and F-actin. The binding of titin motifs to myosin supports the proposed association of A-band titin with the thick filament, and the novel titin-F-actin interaction was confirmed by F-actin cosedimentation assays, suggesting that titin may also be involved in the structure and/or function of the thin filament.

FOOTNOTES

*

This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Alberta. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L38717[GenBank].

§

Recipient of a research scholarship from the Heart and Stroke Foundation of Canada. To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Calgary Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-8861; Fax: 403-270-2211; jjin{at}acs.ucalgary.ca.

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The abbreviations are: PCR, polymerase chain reaction; ABTS, 2,2`-azinobis-(3-ethybenzthiazolinesulfonic acid); B, 50% maximal binding; bp, base pair(s); BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl-1-thio--D-galactopyranoside; mAb, monoclonal antibody; NEPHGE, non-equilibrium pH gradient gel electrophoresis; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

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J.-P. Jin and K. Wang, unpublished results.

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J.-P. Jin, unpublished results.

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