Synthesis and folding of native collagen III model peptides

Solid-phase synthesis of triple-helical peptides, including native collagen III sequences, was started with a trimeric branch, based upon the lysine d ipeptide [Fields, C. G., Mickelson, D. J., Drake, S. L., McCarthy, J. B., a nd Fields, G. B. (1993) J. Biol. Chern. 268, 14153-14160]. Branch synthesis was modified, using TentaGel R as resin, p-hydroxybenzyl alcohol (HMP) as linker, Dde as N-t-protective group, and HATU/HOAT as coupling reagent. Thr ee homotrimeric sequences, each containing the Gly 606-Gly 618 portion of h uman type III collagen, were added to the amino functions of the branch. Th e final incorporation of GlyProHyp triplets, stabilizing the collagen III t riple helix, was performed by using protected GlyProHyp tripeptides, each c ontaining tert-butylated hydroxyproline [P*(tBu)] instead of hydroxyproline (P*). Among the protected tripeptides FmocP*(tBu)PG, FmocPP*(tBu)G, and Fm ocGPP*-(tBu), prepared manually on a chlorotrityl resin, incorporation of F mocPP*(tBu)Gly was best suited for synthesis of large and stable peptides, such as PP*G(8), containing 8 (PP*G)(3) trimers (115 residues, 10610 Da). T he structures of five peptides, differing from each other by the type and n umber of the triplets incorporated, were verified by MALDI-TOF-MS. Their co nformations and thermodynamic data were studied by circular dichroism and d ifferential scanning calorimetry. Except for P*PG(8), containing 8 (P*PG)3 trimers with hydroxyproline in the X position and adopting a polyproline II structure, all peptides were triple-helical in 0.1 M acetic acid and their thermal stabilities ranged from t(1/2) = 39.4 to t(1/2) = 62.5 degrees C, depending on the identity and number of the triplets used. Similar values o f the van't Hoff enthalpy, Delta H-vH, derived from melting curves, and the calorimetric enthalpy, Delta H-cal, obtained from heat capacity curves, in dicate a cooperative ratio of CR = Delta H-vH/Delta H-cal = 1, establishing a two-state process for unfolding of THP(III) peptides. The independence o f the transition temperatures tin on peptide concentration as well as equil ibrium centrifugation data indicate monomolecular dimer(f) to dimer(u) (F-2 < - > U-2) transitions and, in addition, bimolecular dimerf to monomer, tr ansitions (F-2 < - > 2U). The dominance of the concentration-independent mo nomolecular reaction over the concentration-dependent bimolecular reaction makes thermal unfolding of THP(III) peptides appear to be monomolecular. If one designates the molecularity described by the term pseudomonomolecular, unfolding of the dimeric peptides PP*G(6-8) follows a two-state, pseudomon omolecular reaction.