DISSECTING TITIN INTO ITS STRUCTURAL MOTIFS - IDENTIFICATION OF AN ALPHA-HELIX MOTIF NEAR THE TITIN N-TERMINUS

Titin, also known as connectin, is a giant modular protein specificall y found in vertebrate striated muscle. Since the huge size of titin do es not allow a direct structure determination, we have started a long- term project to characterize the protein by cutting it into smaller do mains or structural units. The major part of the titin sequence is ass embled by modules approximate to 100 amino acids long that belong to t wo major protein superfamilies. Most of these modules are linked toget her by stretches of variable length with unique sequence. No direct st ructural characterization has been achieved so far for any of these Li nkers. We present here a study of a stretch located in the titin N-ter minus and part of a linker between two modules. Our attention was draw n toward this region because it shows 100% probability to form a coile d coil when analyzed by a prediction program. A synthetic 38 amino aci d peptide spanning such a sequence was studied in aqueous solution by circular dichroism, nuclear magnetic resonance, and analytical ultrace ntrifugation at various pH, salt, and peptide concentrations. Under al l conditions, it shows a strong tendency to form alpha-helical structu res. In the presence of salt, this conformation is associated with the formation of helical bundles below pH 5. Above pH 5, any aggregate br eaks, and the titin peptide is a monomeric helix in equilibrium with i ts random coil conformation. We discuss the factors which stabilize th e helical conformation and the possible role of this stretch in vivo.