Immunoglobulin-type domains of titin: same fold, different stability?

Titin is a 3-MDa protein thought to form a fibrous intracellular system in vertebrate striated muscle and to play an important role in sarcomere alignment during muscle contraction. It has also been implicated as a "molecular ruler", regulating the assembly and the precise length of the thick filaments [Whiting, A. J., Wardale, J., & Trinick, J. (1989) J. Mol. Biol. 205, 163-169]. Partial sequencing of titin-encoding cDNAs suggests that the protein is organized in a modular fashion, containing two classes of approximately 100-residue repeats [Labeit, S., Barlow, D. P., Gautel, M., Gibson, T., Holt, J., Hsieh, C. L., Francke, U., Leonard, K., Wardale, J., Whiting, A., & Trinick, J. (1990) Nature 345, 273-276]. These motifs, referred to as type I and type II modules, show sequence homology to the fibronectin III and immunoglobulin C2 superfamilies, respectively. Since the type II modules represent the most widely occurring motifs along the titin molecule, we expressed in Escherichia coli three domains of this type spanning different regions of the sarcomere (A-band and M-line) and studied their structure and stability. Using circular dichroism, nuclear magnetic resonance, and fluorescence spectroscopy, we showed that all the fragments examined are independently folded in solution and possess a beta-sheet conformation. Furthermore, employing NMR analysis, we identified an overall folding pattern present in all modules and related to the Ig fold, as previously suggested by theoretical predictions. The stability of the modules over a range of conditions was investigated by measuring key thermodynamic parameters for both thermal and chemical denaturation and by monitoring amide proton exchange as a function of time.(ABSTRACT TRUNCATED AT 250 WORDS)