Mutations resulting in replacement of one obligate Gly residue within the repeating (Gly-Xaa-Yaa) n triplet pattern of the collagen type I triple helix are the major cause of osteogenesis imperfecta (OI). Phenotypes of OI involve fragile bones and range from mild to perinatal lethal. In this study, host-guest triple-helical peptides of the form acetyl-(Gly-Pro-Hyp) 3-Zaa-Pro-Hyp-(Gly-Pro-Hyp)4-GlyGly-amide are used to isolate the influence of the residue replacing Gly on triple-helix stability, with Zaa ‫؍‬ Gly, Ala, Arg, Asp, Glu, Cys, Ser, or Val. Any substitution for Zaa ‫؍‬ Gly (melting temperature, Tm ‫؍‬ 45°C) results in a dramatic destabilization of the triple helix. For Ala and Ser, T m decreases to Ϸ10°C, and for the Arg-, Val-, Glu-, and Asp-containing peptides, Tm < 0°C. A Gly 3 Cys replacement results in T m < 0°C under reducing conditions but shows a broad transition (T m Ϸ 19°C) in an oxidizing environment. Addition of trimethylamine N-oxide increases T m by Ϸ5°C per 1 M trimethylamine N-oxide, resulting in stable triple-helix formation for all peptides and allowing comparison of relative stabilities. The order of disruption of different Gly replacements in these peptides can be represented as Ala < Ser < CPOred < Arg < Val < Glu < Asp. The rank of destabilization of substitutions for Gly in these Gly-ProHyp-rich homotrimeric peptides shows a significant correlation with the severity of natural OI mutations in the ␣1 chain of type I collagen.

The refolding of thermally denatured model collagenlike peptides was studied for a set of 21 guest triplets embedded in a common host framework: acetyl-(GlyPro-Hyp) 3 -Gly-Xaa-Yaa-(Gly-Pro-Hyp) 4 -Gly-Gly-amide. The results show a strong dependence of the folding rate on the identity of the guest Gly-Xaa-Yaa triplet, with the half-times for refolding varying from 6 to 110 min (concentration ‫؍‬ 1 mg/ml). All triplets of the form Gly-XaaHyp promoted rapid folding, with the rate only marginally dependent on the residue in the Xaa position. In contrast, triplets of the form Gly-Pro-Yaa and Gly-XaaYaa were slower and showed a wide range of half-times, varying with the identity of the residues in the triplet. At low concentrations, the folding can be described by third-order kinetics, suggesting nucleation is rate-limiting. Data on the relative nucleation ability of different Gly-Xaa-Yaa triplets support the favorable nature of imino acids, the importance of hydroxyproline, the varying effects of the same residue in the Xaa position versus the Yaa position, and the difficulties encountered when leucine or aspartic acid are in the Yaa position. Information on the relative propensities of different tripeptide sequences to promote nucleation of the triple-helix in peptides will aid in identification of nucleation sites in collagen sequences.The collagen triple helix is the basic structural motif found in all fibril-forming collagens as well as some host-defense proteins such as C1q, mannose-binding protein, and macrophage scavenger receptor (1, 2). The triple-helix conformation consists of three extended polyproline II-like chains supercoiled around each other as determined by x-ray fiber diffraction, crystallography, and NMR (3-7). The three chains are staggered by one residue with respect to each other and stabilized by interchain hydrogen bonding (5,8,9). This conformation requires that every third residue must be a glycine, generating a repeating (Gly-Xaa-Yaa) n pattern, and that a high proportion of residues are the imino acids proline and hydroxyproline. Gly-Pro-Hyp is indeed the most common and stabilizing tripeptide found in collagens. 1The folding of the collagen triple helix in vivo is a multistep process involving chain association, registration, nucleation, and propagation (10 -12). There is also evidence for the involvement of chaperones (13,14). Fibril-forming collagens are synthesized at the rough endoplasmic reticulum membrane in a precursor form, procollagen, containing both N-and C-terminal propeptides terminating the long central triple helix. Proper chain selection and registration is initiated by the association of the C-propeptide domains into trimers followed by nucleation of the correctly aligned triple helix (15, 16) and propagation in a C-to N-terminal direction (17). In the unfolded state, most proline residues in the Yaa position of Gly-Xaa-Yaa triplets are enzymatically hydroxylated, and the resulting hydroxyproline (Hyp) residues are required for the formation and stabilization of the tripl...