Type I collagen triple helix destabilization caused by glycine and arginine mutations.

<p>A, DSC denaturation thermograms of pepsin-purified collagen (solid lines) and procollagen (dotted lines) secreted into media by normal control (NC) and patient cells. B, Denaturation thermograms of pepsin-purified collagen from media (secreted, solid lines), cell layer (intracellular, dashed line), and extracellular matrix deposited by cells (matrix, dash-dotted lines). C,D, Analysis of α1(I)-R780C collagen thermal stability by enzymatic digestion and gel electrophoresis. In A and B, each thermogram peak represents denaturation of molecules with or without mutant chains. The peak maximum is the corresponding apparent denaturation temperature T_m, which is the same in collagen and procollagen molecules (for NC as well as most mutants) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200264#pone.0200264.ref027" target="_blank">27</a>]. A heterozygous Gly substitution in the α1(I) chain might result in up to 3 peaks on the denaturation thermogram, representing molecules with 2, 1, and 0 mutant chains. In α1(I)-G766C collagen, the molecules with 1 and 2 mutant chains have the same T_m, producing one peak at ≈39.5 °C; the molecules without mutant chains produce the second peak at ≈42 °C. The same is true for α1(I)-G763S collagen. In α1(I)-R780L or α1(I)-R780C, molecules with 1 and 2 mutant chains produce a peak at ≈41 °C; the 42 °C peak of molecules without the mutant chains is too close to 41 °C, so that it is not resolved. The area under each peak is proportional to the fraction of the corresponding molecules. A change in this fraction upon secretion from cells or incorporation into matrix alters the shape of the denaturation thermogram [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200264#pone.0200264.ref027" target="_blank">27</a>]. A reduced intensity of the 39.5 °C peak in matrix vs. secreted collagen indicated that only 25% of α1(I)-G766C molecules in extracellular matrix contained mutant chains, but no effects of other mutations on collagen secretion or matrix incorporation were detected. In C and D, thermal stability of secreted collagen was measured by 2 min equilibration at different temperatures followed by 5 min at room temperature, 1 min digestion with trypsin/chymotrypsin mixture, and separation of chains by gel electrophoresis at non-reducing conditions as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200264#pone.0200264.ref031" target="_blank">31</a>]. Panel C shows quantitative analysis of gel electrophoresis (D), each point representing an average of 4 experiments.