Native collagen has a two-bonded structure

Abstract

A number of structural models that differ sterically and in interpeptide hydrogen-bonding pattern have been put forward for the native collagen molecule. These have proven remarkably difficult to distinguish by X-ray diffraction and other methods. The various models have generally been divided into a “one-bonded” class (one hydrogen bond per triad of amino acid residues) and a “two-bonded” class (nearly two bonds per triad). To discriminate between the models, we have applied hydrogen-tritium exchange methods to native soluble ichthyocol (carp swim bladder) collagen. Free and hydrogen-bonded peptide groups were recognized by their hydrogen exchange rates. Under two different solution conditions, a set of peptide hydrogens was found to display exchange rates nearly identical to those previously calibrated for free peptides in model systems and, therefore, was taken to represent free peptides. The remaining hydrogens exchanged much more slowly and were assigned to internally hydrogen-bonded peptide groups. The numbers of free and of internally bonded peptide hydrogens were measured independently in different kinds of exchange experiments. They coincide with the numeration required for two-bonded models. The results appear to exclude one-bonded models for ichthyocol collagen but cannot distinguish between the earlier two-bonded model, which contains two direct peptide to peptide hydrogen bonds per triad, and the more recent proposal involving one direct bond and one cross-bridging water molecule. The results, together with other data in the literature, indicate that native collagens from other species may also exist in a two-bonded structure, though some synthetic sequence polypeptides related to collagen do clearly assume a one-bonded structure. The results are relevant also to the general problem of structure analysis by hydrogen exchange methods. It has previously been demonstrated that free peptide hydrogens exchange at precisely predictable rates in small molecules, in oligo- and polypeptides, and in a globular protein. The present data extend this conclusion to a fibrous protein. Thus the techniques used here may be quite generally applied to distinguish and to count free and internally bonded peptide groups in protein and polypeptide systems.