Interacting Sites in Novel Polymeric Proteins.

The object of this project was the folding and association of Cys-rich recombinant and native silk proteins from aquatic larvae of the midge, Chironomus tentans. Specific aims include. (A) Learn which Cys residues are critical to the pathway for intramolecular disulfide bond formation in rCAS, a recombinant repeat from silk protein, spIa. (B) Determine if intramolecular disulfide bonds stabilize the higher-order structure of rCAS. (C) Determine the conditions and which Cys participate in formation of intermolecular disulfide bonds. (D) Learn how and where other native silk proteins interact with spIs. In addition, we also chose to do the following. (E) Determine if N-linked glycosylation sites in ssp160 are evolutionarily conserved. (F) Acquire consensus repeat sequences for Cys-containing motifs in sp185/sp220 homologs and sp195. The final results of this project indicate: (1) Aquatic silk proteins can be expressed and purified from bacteria in large quantities. (2) However, fibrous proteins may prove difficult to analyze: CD and FTTR basis spectra lack fibrous protein standards and proteins with Cys may refold into multiple conformers. (3) The abundance and conservation of glycosylation sites on some aquatic silk proteins suggest such post-translational modification merits study before biotechnological applications are considered. (4) Aquatic silk proteins contain more Cys motifs than previously thought.