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 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 was modified, using TentaGel R as resin, p-hydroxybenzyl alcohol (HMP) as linker, Dde as N(epsilon)-protective group, and HATU/HOAT as coupling reagent. Three homotrimeric sequences, each containing the Gly 606-Gly 618 portion of human type III collagen, were added to the amino functions of the branch. The final incorporation of GlyProHyp triplets, stabilizing the collagen III triple helix, was performed by using protected GlyProHyp tripeptides, each containing tert-butylated hydroxyproline [P(tBu)] instead of hydroxyproline (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 for synthesis of large and stable peptides, such as PPG(8), containing 8 (PPG)(3) trimers (115 residues, 10 610 Da). The structures of five peptides, differing from each other by the type and number of the triplets incorporated, were verified by MALDI-TOF-MS. Their conformations and thermodynamic data were studied by circular dichroism and differential scanning calorimetry. Except for PPG(8), containing 8 (PPG)(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 of the van't Hoff enthalpy, DeltaH(vH), derived from melting curves, and the calorimetric enthalpy, DeltaH(cal), obtained from heat capacity curves, indicate a cooperative ratio of CR = DeltaH(vH)/DeltaH(cal) = 1, establishing a two-state process for unfolding of THP(III) peptides. The independence of the transition temperatures t(1/2) on peptide concentration as well as equilibrium centrifugation data indicate monomolecular dimer(f) to dimer(u) (F(2) <--> U(2)) transitions and, in addition, bimolecular dimer(f) to monomer(u) transitions (F(2) <--> 2U). The dominance of the concentration-independent monomolecular 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 PPG(6-8) follows a two-state, pseudomonomolecular reaction.
Henkel
W
W
Institut für Arterioskleroseforschung an der Universität Münster, Germany. henkelw@uni-muenster.de
Vogl
T
T
Echner
H
H
Voelter
W
W
Urbanke
C
C
Schleuder
D
D
Rauterberg
J
J
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003094:Collagen / Q000138:chemical synthesis* / Q000302:isolation & purification; D005998:Glycine / Q000737:chemistry; D006909:Hydroxyproline / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis* / Q000302:isolation & purification; D011392:Proline / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D014461:Ultracentrifugation
D009842:Oligopeptides; D010455:Peptides; D003094:Collagen; D011392:Proline; D006909:Hydroxyproline; D005998:Glycine