RECOMBINANT PROCOLLAGEN-II - DELETION OF D-PERIOD SEGMENTS IDENTIFIESSEQUENCES THAT ARE REQUIRED FOR HELIX STABILIZATION AND GENERATES A TEMPERATURE-SENSITIVE N-PROTEINASE CLEAVAGE SITE

A cDNA cassette system was used to synthesize recombinant versions of procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were delete d, All the proteins were triple helical and all underwent a helix-to-c oil transition between 25 and 42 degrees C as assayed by circular dich roism, However, the details of the melting curves varied. The procolla gen lacking the DI period unfolded 3 degrees C lower than a full-lengt h molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length mole cule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unf olded 3 degrees C lower than the full-length molecule and a smaller fr action of the protein was secreted by stably transfected clones than w ith the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varyin g stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and f or stabilizing the triple helix. Reaction of the recombinant procollag ens with procollagen N-proteinase indicated that in the procollagen la cking the sequences, the D1 period assumed an unusual temperature-sens itive conformation at 35 degrees C that allowed cleavage at an otherwi se resistant Gly-Ala bond between residues 394 and 395 of the alpha 1( II) chain.