Abstract
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 deleted. All the proteins were triple helical and all underwent a helix-to-coil transition between 25 and 42 degreesC as assayed by circular dichroism. However, the details of the melting curves varied. The procollagen lacking the D1 period unfolded 3 degreesC lower than a full-length molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length molecule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unfolded 3 degreesC lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant procollagens with procollagen N-proteinase indicated that in the procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35 degreesC that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the alpha1(II) chain.
Highlights
The fibrillar collagens are major structural proteins that largely define the size, shape, and strength of tissues in most complex organisms [1,2,3,4]
One of the most direct indications of regions of varying stability in the triple helix came from mutations that convert different obligate glycine codons to codons for amino acids with bulkier side chains and cause the brittleness of bones and other tissues characteristic of the heritable disease known as osteogenesis imperfecta (3, 18 –20)
Since the procollagen II lacking the D1 period unfolded at a lower temperature than the FL molecule, the 234 residues in the D1 period must be rich in sequences that stabilize the triple helix and, serve as an N-terminal clamp for the helix
Summary
The fibrillar collagens are major structural proteins that largely define the size, shape, and strength of tissues in most complex organisms [1,2,3,4]. Evidence for the microunfolding of the monomer included the effects of partially denaturing and renaturing the protein [8, 9], experiments involving reversible inhibition of hydroxylation of proline and lysine residues during biosynthesis [10], comparisons of the helix-forming properties of synthetic peptides with repetitive -Gly-Xxx-Yyy- sequences [11,12,13,14,15,16], measurements of enthalpy changes by microcalorimetry [7], and the effects of temperature on the kinetics of fibril formation [17]. One of the most direct indications of regions of varying stability in the triple helix came from mutations that convert different obligate glycine codons to codons for amino acids with bulkier side chains and cause the brittleness of bones and other tissues characteristic of the heritable disease known as osteogenesis imperfecta (3, 18 –20).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
