TYPE-I COLLAGEN CNBR PEPTIDES - SPECIES AND BEHAVIOR IN SOLUTION

The properties of type I collagen CNBr peptides in solution were studi ed to investigate the molecular species formed, their conformation, an d factors influencing equilibria between peptide species. Peptides for med homologous trimers, even though the native parent protein is heter otrimeric, [alpha 1(I)](2) alpha 2(I). Their triple-helical content wa s 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 appea red to play a minor role in reducing helicity. Circular dichroism spec tra and resistance to trypsin digestion at 4 and 20 degrees C demonstr ated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) C B7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability t o autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecul es were disrupted by 0.5 M NaCl, demonstrating that their ionic charac ter is important. Changes in ionic strength also altered the hydrodyna mic size of single- and triple-stranded molecules. The different molec ular species are in equilibrium. The kinetics of the conversion of tri mer to monomer species was determined in a time course experiment usin g 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 l ower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mo l. The circular dichroism profile at increasing temperatures showed th at the melting temperature for triple-helical peptides is about 6-10 d egrees C lower than that of the parent native type I collagen. The fol ding of peptides is a spontaneous process (exothermic but with unfavou rable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from he at-denatured samples.