Abstract:Collagen-like oligopeptides of defined chain length were synthesized via the following tripeptides which were combined stepwise: Boc-Gly-L-Pro-L-Pro-OH (I), Boc-Gly-L-Pro-L-Lys-OH (2), BOG-L-Ala-Gly-L-Pro-OH (4), Boc-Gly-L-Pro-L-Leu-OH (6), and Boc-L-Ala-L-Hyp(0Bz)-Gly-OH (9). Two methods for covalent bridging of three oligopeptides of the same sequence were applied resulting in the formation ofpolypeptides of type 22 or 23.
EinleitungDas Faserprotein Kollagen durchlauft beim Erwarmen in waBriger Losung einen … Show more
“…Refolding of thermally denatured collagen fragments and collagenous peptides is known to be a very slow process that requires days and does not occur quantitatively in all cases, , whereas for the triple helix formation in collagens and procollagens which contain various types of interchain cross-links half times of 30 min to several hours were measured. , As shown in Figure for the heterotrimer D, after a temperature jump from 53 to 3 °C refolding of the disulfide-bridged heterotrimer at 3 °C is a very fast process consisting of a first kinetically unresolved and a second slower phase with a rate constant of k = 5.6·10 -4 s -1 . A similarly fast refolding in two phases has previously been reported for the Col 1−3 fragment derived from the N-terminal propeptide of pN-collagen III 25 and for synthetic collagenous homotrimers cross-linked with the 6-aminohexanoyl-di-lysine template. , 7 (A) thermal unfolding (▪) and refolding (○) of the heterotrimer B in 50 mM Tris/HCl, pH 7.4 (10 mM CaCl 2 , 50 mM NaCl) as monitored by changes in the dichroic intensity at 222 nm; heating and cooling rates: 0.3 °C/min; (B) CD spectra of the heterotrimer B at 4 °C prior thermal denaturation () and after refolding (···). 8 Kinetics of the triple helix refolding of the heterotrimer D in 50 mM Tris/HCl, pH 7.4 (10 mM CaCl 2 , 50 mM NaCl) after quenching the solution from 53 °C to 3 °C as determined by changes in dichroic intensity at 222 nm. From the semilogarithmic plot of the experimental data against time a first-order rate constant k exp = 5.6·10 -4 s -1 was derived.…”
Section: Resultsmentioning
confidence: 99%
“…A similarly fast refolding in two phases has previously been reported for the Col 1-3 fragment derived from the N-terminal propeptide of pN-collagen III 25 and for synthetic collagenous homotrimers cross-linked with the 6-aminohexanoyl-di-lysine template. 56,57 Thermodynamic Characterization of the Heterotrimeric Collagenous Peptides. Thermal denaturation of natural and synthetic collagenous peptides is generally regarded as a twostate transition.…”
Collagenous peptides containing the collagenase cleavage site α1(772−784) and α2(772−784) of
collagen type I were synthesized and assembled into heterotrimers via regioselective C-terminal interchain-disulfide bridging in a defined α1α2α1‘ staggered register of the three peptide strands. Various approaches
were attempted to induce and stabilize the collagen-characteristic triple-helical fold even in the sequence portion
of the collagenase cleavage site with its weak triple-helix propensity. By N-terminal chain elongation with
(Gly-Pro-Hyp)
n
tripeptide repeats, particularly with n = 5, and in an even more pronounced manner, by
incorporation of an additional tripeptide repeat adjacent to the cystine knot, a collagenous heterotrimer was
obtained which was found to exhibit dichroic properties fully consistent with the triple-helical fold. Thermal
denaturation revealed a remarkable stability with a melting temperature of 41 °C. Although the complex cystine
knot of natural collagen was reduced in these synthetic heterotrimers to two interchain-disulfide bridges, it
showed not only the expected entropic contribution to the refolding process by keeping the three chains
assembled, but more importantly a triple-helix nucleation was induced. In fact, temperature jump experiments
clearly revealed two-phase refolding kinetics very similar to those of the disulfide-bridged natural collagen
fragment of Col 1−3, where refolding without nucleation difficulty was obtained followed by a slower process
dominated by the cis → trans isomerization for triple-helix propagation. These results would indicate that
even the simplified artificial cystine knot is capable of aligning the three peptide chains in the defined α1α2α1‘
one-residue shift register. Moreover, the synthetic heterotrimers were cleaved by interstitial collagenases in a
single cut through all three chains without release of intermediates during the relatively slow enzymatic digestion
process. This observation confirms that, with the de novo designed heterotrimers, functional collagen epitopes
were mimicked in highly efficient manner; it also strongly suggests that the preselected α1α2α1‘ register may
indeed represent the correct staggered alignment of the α subunits at least in collagen type I.
“…Refolding of thermally denatured collagen fragments and collagenous peptides is known to be a very slow process that requires days and does not occur quantitatively in all cases, , whereas for the triple helix formation in collagens and procollagens which contain various types of interchain cross-links half times of 30 min to several hours were measured. , As shown in Figure for the heterotrimer D, after a temperature jump from 53 to 3 °C refolding of the disulfide-bridged heterotrimer at 3 °C is a very fast process consisting of a first kinetically unresolved and a second slower phase with a rate constant of k = 5.6·10 -4 s -1 . A similarly fast refolding in two phases has previously been reported for the Col 1−3 fragment derived from the N-terminal propeptide of pN-collagen III 25 and for synthetic collagenous homotrimers cross-linked with the 6-aminohexanoyl-di-lysine template. , 7 (A) thermal unfolding (▪) and refolding (○) of the heterotrimer B in 50 mM Tris/HCl, pH 7.4 (10 mM CaCl 2 , 50 mM NaCl) as monitored by changes in the dichroic intensity at 222 nm; heating and cooling rates: 0.3 °C/min; (B) CD spectra of the heterotrimer B at 4 °C prior thermal denaturation () and after refolding (···). 8 Kinetics of the triple helix refolding of the heterotrimer D in 50 mM Tris/HCl, pH 7.4 (10 mM CaCl 2 , 50 mM NaCl) after quenching the solution from 53 °C to 3 °C as determined by changes in dichroic intensity at 222 nm. From the semilogarithmic plot of the experimental data against time a first-order rate constant k exp = 5.6·10 -4 s -1 was derived.…”
Section: Resultsmentioning
confidence: 99%
“…A similarly fast refolding in two phases has previously been reported for the Col 1-3 fragment derived from the N-terminal propeptide of pN-collagen III 25 and for synthetic collagenous homotrimers cross-linked with the 6-aminohexanoyl-di-lysine template. 56,57 Thermodynamic Characterization of the Heterotrimeric Collagenous Peptides. Thermal denaturation of natural and synthetic collagenous peptides is generally regarded as a twostate transition.…”
Collagenous peptides containing the collagenase cleavage site α1(772−784) and α2(772−784) of
collagen type I were synthesized and assembled into heterotrimers via regioselective C-terminal interchain-disulfide bridging in a defined α1α2α1‘ staggered register of the three peptide strands. Various approaches
were attempted to induce and stabilize the collagen-characteristic triple-helical fold even in the sequence portion
of the collagenase cleavage site with its weak triple-helix propensity. By N-terminal chain elongation with
(Gly-Pro-Hyp)
n
tripeptide repeats, particularly with n = 5, and in an even more pronounced manner, by
incorporation of an additional tripeptide repeat adjacent to the cystine knot, a collagenous heterotrimer was
obtained which was found to exhibit dichroic properties fully consistent with the triple-helical fold. Thermal
denaturation revealed a remarkable stability with a melting temperature of 41 °C. Although the complex cystine
knot of natural collagen was reduced in these synthetic heterotrimers to two interchain-disulfide bridges, it
showed not only the expected entropic contribution to the refolding process by keeping the three chains
assembled, but more importantly a triple-helix nucleation was induced. In fact, temperature jump experiments
clearly revealed two-phase refolding kinetics very similar to those of the disulfide-bridged natural collagen
fragment of Col 1−3, where refolding without nucleation difficulty was obtained followed by a slower process
dominated by the cis → trans isomerization for triple-helix propagation. These results would indicate that
even the simplified artificial cystine knot is capable of aligning the three peptide chains in the defined α1α2α1‘
one-residue shift register. Moreover, the synthetic heterotrimers were cleaved by interstitial collagenases in a
single cut through all three chains without release of intermediates during the relatively slow enzymatic digestion
process. This observation confirms that, with the de novo designed heterotrimers, functional collagen epitopes
were mimicked in highly efficient manner; it also strongly suggests that the preselected α1α2α1‘ register may
indeed represent the correct staggered alignment of the α subunits at least in collagen type I.
“…To overcome the unfavorable entropy of nucleating the triple helix in synthetic collagen-like peptides and to increase the thermal stability of the super-helix, covalent linkage of the peptide chains to templates such as 1,2,3-propanetricarboxylic acid or the Lys-Lys dipeptide, has been proposed [10][11][12][13]. The Lys-Lys template approach has extensively been exploited in the solid-phase synthesis of homotrimeric collagenous peptides [14] and even of a heterotrimer [15], despite the serious difficulties encountered in the synthesis and purification of such high molecular weight polypeptides.…”
A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site (P4-P'+/t0) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp)s triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triplestranded cystine peptide. While the single chains showed no teodeney to self-association into triple helices, the heterotrimer (cxltz2~xl') was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature (Tin) of
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
