Structural properties of recombinant nonrepetitive and repetitive parts of major ampullate spidroin 1 from Euprosthenops australis : implications for fiber formation

• Spider dragline silk proteins, spidroins, have a tripartite composition; a nonrepetitive N-terminal domain, a central repetitive region built up from many iterated poly-Ala and Gly rich blocks, and a C-terminal nonrepetitive domain. It is generally believed that the repetitive region forms intermolecular contacts in the silk fibers, while precise functions of the terminal domains have not been established. Herein, thermal, pH, and salt effects on the structure and aggregation and/or polymerization of recombinant N- and C-terminal domains, a repetitive segment containing four poly-Ala and Gly rich coblocks, and combinations thereof were studied. The N- and C-terminal domains have mainly alpha-helical structure, and interestingly, both form homodimers. Dimerization of the end domains allows spidroin multimerization independent of the repetitive part. Reduction of an intersubunit disulfide in the C-terminal domain lowers the thermal stability but does not affect dimerization. The repetitive region shows helical secondary structure but appears to lack stable folded structure. A protein composed of this repetitive region linked to the C-terminal domain has a mainly alpha-helical folded structure but shows an abrupt transition to beta-sheet structures upon heating. At room temperature, this protein self-assembles into macroscopic fibers within minutes. The secondary structures of none of the domains are altered by pH or salt. However, high concentrations of phosphate affect the tertiary structure and accelerate the aggregation propensity of the repetitive region. Implications of these results for dragline spidroin behavior in solution and silk fiber formation are discussed.

Nyckelord

Amino Acid Sequence

Animals

Fibroins/biosynthesis/*chemistry

Molecular Sequence Data

Protein Conformation

Protein Structure

Tertiary

Recombinant Fusion Proteins/biosynthesis/*chemistry

*Repetitive Sequences

Amino Acid

Spiders/*chemistry

Temperature

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)