Collagen triple helix formation can be nucleated at either end

Abstract

The directional dependence of folding rates for rodlike macromolecules such as parallel α-helical coiled-coils, DNA double-helices, and collagen triple helices is largely unexplored. This is mainly due to technical difficulties in measuring rates in different directions. Folding of collagens is nucleated by trimeric non-collagenous domains. These are usually located at the COOH terminus, suggesting that triple helix folding proceeds from the COOH to the NH2 terminus. Evidence is presented here that effective nucleation is possible at both ends of the collagen-like peptide (Gly-Pro-Pro)10, using designed proteins in which this peptide is fused either NH2- or COOH-terminal to a nucleation domain, either T4-phage foldon or the disulfide knot of type III collagen. The location of the nucleation domain influences triple-helical stability, which might be explained by differences in the linker sequences and the presence or absence of repulsive charges at the carboxyl-terminal end of the triple helix. Triple helical folding rates are found to be independent of the site of nucleation and consistent with cis-trans isomerization being the rate-limiting step.