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 specifically found in vertebrate striated muscle. Since the huge size of titin does not allow a direct structure determination, we have started a long-term project to characterize the protein by cutting it into smaller domains or structural units. The major part of the titin sequence is assembled by modules approximately 100 amino acids long that belong to two major protein superfamilies. Most of these modules are linked together by stretches of variable length with unique sequence. No direct structural characterization has been achieved so far for any of these linkers. We present here a study of a stretch located in the titin N-terminus and part of a linker between two modules. Our attention was drawn toward this region because it shows 100% probability to form a coiled coil when analyzed by a prediction program. A synthetic 38 amino acid peptide spanning such a sequence was studied in aqueous solution by circular dichroism, nuclear magnetic resonance, and analytical ultracentrifugation at various pH, salt, and peptide concentrations. Under all conditions, it shows a strong tendency to form alpha-helical structures. In the presence of salt, this conformation is associated with the formation of helical bundles below pH 5. Above pH 5, any aggregate breaks, and the titin peptide is a monomeric helix in equilibrium with its random coil conformation. We discuss the factors which stabilize the helical conformation and the possible role of this stretch in vivo.
Musco
G
G
EMBL, Heidelberg, Germany.
Tziatzios
C
C
Schuck
P
P
Pastore
A
A
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D064211:Connectin; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011494:Protein Kinases; D012492:Salts; D012996:Solutions; D014461:Ultracentrifugation; D014867:Water
D064211:Connectin; D009124:Muscle Proteins; D010446:Peptide Fragments; D012492:Salts; D012996:Solutions; D014867:Water; D011494:Protein Kinases