Site-specific NMR monitoring of cis-trans isomerization in the folding of the proline-rich collagen triple helix

Understanding the folding of the proline-rich collagen triple helix require s consideration of the effects of proline cis-trans isomerization and may s hed light on the misfolding of collagen in connective tissue diseases, Fold ing was monitored in real time by heteronuclear 2D NMR spectroscopy for the N-15 labeled positions in the triple-helical peptide T1-892 [GPAGPAGPVGPAG ARGPAGPOGPOGPOG POGV]. In the equilibrium unfolded monomer form, each label ed residue showed multiple peaks with interconversion rates consistent with cis-trans isomerization of Gly-Pro and Pro-Hyp bonds. Realtime NMR studies on the folding of T1-892 showed slow decay of monomer peaks and a concomit ant increase in trimer peaks. Gly25 in the C-terminal rich (Gly-Pro-Hyp)(4) domain folds first, consistent with its being a nucleation domain. Analysi s of the kinetics indicates that the folding of Gly25 is biphasic and the s lower step represents cis-trans isomerization of imino acids. This illustra tes that nucleation is limited by cis-trans isomerization. Monitoring Gly6, Gly10, Ala12, and Gly13 monomer and trimer peaks captures the C- to N-term inal propagation of the triple helix, which is also limited by Gly-Pro cis- trans isomerization events. The zipper-like nature of the propagation proce ss is confirmed by the slower rate of folding of Ala6 compared to Gly13, re flecting the larger number of isomerization events encountered by the more N-terminal Ala6. The cis-trans isomerization events at multiple proline res idues is a complex statistical process which can be visualized by these NMR studies.