Synthesis and Folding of Native Collagen III Model Peptides
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- 作者：Werner Henkel ; Thomas Vogl ; Hartmut Echner ; Wolfgang Voelter ; Claus Urbanke ; Detlef Schleuder ; and Jü ; rgen Rauterberg
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：October 12, 1999
- 年：1999
- 卷：38
- 期：41
- 页码：13610 - 13622
- 全文大小：195K
- 年卷期：v.38,no.41(October 12, 1999)
- ISSN：1520-4995
文摘
Solid-phase synthesis of triple-helical peptides, including native collagen III sequences, wasstarted with a trimeric branch, based upon the lysine dipeptide [Fields, C. G., Mickelson, D. J., Drake, S.L., McCarthy, J. B., and Fields, G. B. (1993) J. Biol. Chem. 268, 14153-14160]. Branch synthesis wasmodified, using TentaGel R as resin, p-hydroxybenzyl alcohol (HMP) as linker, Dde as N
-protectivegroup, and HATU/HOAT as coupling reagent. Three homotrimeric sequences, each containing the Gly606-Gly 618 portion of human type III collagen, were added to the amino functions of the branch. Thefinal incorporation of GlyProHyp triplets, stabilizing the collagen III triple helix, was performed by usingprotected GlyProHyp tripeptides, each containing tert-butylated hydroxyproline [P*(tBu)] instead ofhydroxyproline (P*). Among the protected tripeptides FmocP*(tBu)PG, FmocPP*(tBu)G, and FmocGPP*(tBu), prepared manually on a chlorotrityl resin, incorporation of FmocPP*(tBu)Gly was best suited forsynthesis of large and stable peptides, such as PP*G(8), containing 8 (PP*G)3 trimers (115 residues,10 610 Da). The structures of five peptides, differing from each other by the type and number of thetriplets incorporated, were verified by MALDI-TOF-MS. Their conformations and thermodynamic datawere studied by circular dichroism and differential scanning calorimetry. Except for P*PG(8), containing8 (P*PG)3 trimers with hydroxyproline in the X position and adopting a polyproline II structure, all peptideswere triple-helical in 0.1 M acetic acid and their thermal stabilities ranged from t1/2 = 39.4 to t1/2 = 62.5
C, depending on the identity and number of the triplets used. Similar values of the van't Hoff enthalpy,
HvH, derived from melting curves, and the calorimetric enthalpy, Hcal, obtained from heat capacitycurves, indicate a cooperative ratio of CR = HvH/Hcal = 1, establishing a two-state process for unfoldingof THP(III) peptides. The independence of the transition temperatures t1/2 on peptide concentration aswell as equilibrium centrifugation data indicate monomolecular dimerf to dimeru (F2 
U2) transitionsand, in addition, bimolecular dimerf to monomeru transitions (F2 2U). The dominance of theconcentration-independent monomolecular reaction over the concentration-dependent bimolecular reactionmakes thermal unfolding of THP(III) peptides appear to be monomolecular. If one designates themolecularity described by the term pseudomonomolecular, unfolding of the dimeric peptides PP*G(6-8)follows a two-state, pseudomonomolecular reaction.
-protectivegroup, and HATU/HOAT as coupling reagent. Three homotrimeric sequences, each containing the Gly606-Gly 618 portion of human type III collagen, were added to the amino functions of the branch. Thefinal incorporation of GlyProHyp triplets, stabilizing the collagen III triple helix, was performed by usingprotected GlyProHyp tripeptides, each containing tert-butylated hydroxyproline [P*(tBu)] instead ofhydroxyproline (P*). Among the protected tripeptides FmocP*(tBu)PG, FmocPP*(tBu)G, and FmocGPP*(tBu), prepared manually on a chlorotrityl resin, incorporation of FmocPP*(tBu)Gly was best suited forsynthesis of large and stable peptides, such as PP*G(8), containing 8 (PP*G)3 trimers (115 residues,10 610 Da). The structures of five peptides, differing from each other by the type and number of thetriplets incorporated, were verified by MALDI-TOF-MS. Their conformations and thermodynamic datawere studied by circular dichroism and differential scanning calorimetry. Except for P*PG(8), containing8 (P*PG)3 trimers with hydroxyproline in the X position and adopting a polyproline II structure, all peptideswere triple-helical in 0.1 M acetic acid and their thermal stabilities ranged from t1/2 = 39.4 to t1/2 = 62.5C, depending on the identity and number of the triplets used. Similar values of the van't Hoff enthalpy,HvH, derived from melting curves, and the calorimetric enthalpy, Hcal, obtained from heat capacitycurves, indicate a cooperative ratio of CR = HvH/Hcal = 1, establishing a two-state process for unfoldingof THP(III) peptides. The independence of the transition temperatures t1/2 on peptide concentration aswell as equilibrium centrifugation data indicate monomolecular dimerf to dimeru (F2 U2) transitionsand, in addition, bimolecular dimerf to monomeru transitions (F2 2U). The dominance of theconcentration-independent monomolecular reaction over the concentration-dependent bimolecular reactionmakes thermal unfolding of THP(III) peptides appear to be monomolecular. If one designates themolecularity described by the term pseudomonomolecular, unfolding of the dimeric peptides PP*G(6-8)follows a two-state, pseudomonomolecular reaction.