A C-13 NMR-STUDY ON COLLAGENS IN THE SOLID-STATE - HYDRATION DEHYDRATION-INDUCED CONFORMATIONAL CHANGE OF COLLAGEN AND DETECTION OF INTERNAL MOTIONS

We recorded C-13 NMR spectra of type I and IV collagens in the anhydrous and hydrated states, in order to confirm our previous assignment of peaks, and to analyze the mode of partial renaturation of soluble collagens by hydration, as well as rapid intramolecular motions such as ring puckering in proline or hydroxyproline residues. First, we attempted to assign all C-13 NMR peaks of collagen fibrils on the basis of computer simulation by utilizing amino-acid composition and chemical shift data from both the solid state and solution. We confirmed that some previously unassigned peaks were not ascribable to a denatured portion but to the minor amino-acid residues. The C-13 NMR peaks from soluble collagens were appreciably broadened and some peaks were displaced as compared with those of intact collagen fibrils. This was caused by the presence of a partial conformational disorder and/or denaturation at the time of acid-solubilization and dehydration. Those line broadening and displacements of peaks, however, were partially removed by humidification under an atmosphere of 96% R.H. over 12 h. Furthermore, we found that the C-13 spin-lattice relaxation times (T1s) of both the C(beta) and C(gamma) carbons of Pro and Hyp in fibrils are substantially reduced as compared with those of some crystalline oligopeptides. It was shown that the presence of rapid ring puckering motion in these residues results in a reduction of the NT1 values, where N stands for the number of protons attached to the carbon under consideration. It is noteworthy that the order of NT1-gamma < NT1-beta < NT1-delta < NT1-alpha for Pro and Hyp in collagen, collagen-like polypeptides, and some oligopeptides is opposite to that obtained in aqueous solution.