The properties of type I collagen CNBr peptides in solution were studied to investigate the molecular species formed, their conformation, and factors influencing equilibria between peptide species. Peptides formed homologous trimers, even though the native parent protein is heterotrimeric, [alpha 1(I)]2 alpha 2-(I). Their triple-helical content was found to be high (> 75% for most peptides). Full helical content was not reached mainly because of the presence of monomer species; chain misalignment, if present, and trimer unraveling at terminal ends appeared to play a minor role in reducing helicity. Circular dichroism spectra and resistance to trypsin digestion at 4 and 20 degrees C demonstrated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) CB7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability to autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecules were disrupted by 0.5 M NaCl, demonstrating that their ionic character is important. Changes in ionic strength also altered the hydrodynamic size of single- and triple-stranded molecules. The different molecular species are in equilibrium. The kinetics of the conversion of trimer to monomer species was determined in a time course experiment using trypsin digestion and found to be a relatively slow process (trimer half-life is a few days at 4 degrees C, about one order of magnitude lower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mol. The circular dichroism profile at increasing temperatures showed that the melting temperature for triple-helical peptides is about 6-10 degrees C lower than that of the parent native type I collagen. The folding of peptides is a spontaneous process (exothermic but with unfavourable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from heat-denatured samples.
Type I collagen CNBr peptides: species and behavior in solution
ROSSI, ANTONIO;MONZANI, ENRICO;CETTA, GIUSEPPE;TENNI, RUGGERO
1996-01-01
Abstract
The properties of type I collagen CNBr peptides in solution were studied to investigate the molecular species formed, their conformation, and factors influencing equilibria between peptide species. Peptides formed homologous trimers, even though the native parent protein is heterotrimeric, [alpha 1(I)]2 alpha 2-(I). Their triple-helical content was found to be high (> 75% for most peptides). Full helical content was not reached mainly because of the presence of monomer species; chain misalignment, if present, and trimer unraveling at terminal ends appeared to play a minor role in reducing helicity. Circular dichroism spectra and resistance to trypsin digestion at 4 and 20 degrees C demonstrated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) CB7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability to autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecules were disrupted by 0.5 M NaCl, demonstrating that their ionic character is important. Changes in ionic strength also altered the hydrodynamic size of single- and triple-stranded molecules. The different molecular species are in equilibrium. The kinetics of the conversion of trimer to monomer species was determined in a time course experiment using trypsin digestion and found to be a relatively slow process (trimer half-life is a few days at 4 degrees C, about one order of magnitude lower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mol. The circular dichroism profile at increasing temperatures showed that the melting temperature for triple-helical peptides is about 6-10 degrees C lower than that of the parent native type I collagen. The folding of peptides is a spontaneous process (exothermic but with unfavourable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from heat-denatured samples.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
