Formula omitted] high power double magnetic resonance investigation of collagen backbone motion in fibrils and in solution

Abstract

13C 1H high power double magnetic resonance spectroscopy was used to investigate the mobility of the collagen peptide backbone. [1- 13C]- and [2- 13C]-glycine-labeled collagen samples (with >50% enrichment in 13C) were prepared via chick calvaria culture. 13C n.m.r. † † Abbreviations used: n.m.r., nuclear magnetic resonance; p.p.m., parts per million; NOE, nuclear Overhauser enhancement. spectra of labeled reconstituted collagen fibrils, of labeled helical collagen in solution, and of unlabeled bovine Achilles tendon collagen were obtained with scalar decoupling and with dipolar decoupling of protons. Proton-enhanced spectra were also obtained using cross-polarization techniques. n.m.r. parameters (linewidths, lineshapes, T 1 values, nuclear Overhauser enhancements, and cross polarization enhancements) were measured for the labeled samples and for collagen in natural abundance. Comparison of 13C n.m.r. parameters for bovine Achilles tendon fibrils and for reconstituted chick calvaria collagen fibrils established that chick calvaria collagen is a good model for the molecular dynamics of collagen in vivo. Spin-lattice relaxation times and nuclear Overhauser enhancements for [1- 13C]- and [2- 13C]glycine-labeled collagen indicated that R 1 ~2 × 10 7 s −1 in solution, where R 1 is the diffusion constant for reorientation about the long axis of the molecule. A substantially smaller value for R 1 (2.6 × 10 6 s −1) was calculated for an axially symmetric ellipsoid of revolution having dimensions appropriate to the collagen helix. The discrepancy between the rigid ellipsoid and n.m.r. values of R 1 suggests that the collagen molecule undergoes torsional reorientation, as well as rod-like reorientation, about its long axis. The T 1 and NOE values measured in the glycine-labeled fibrils show that rapid axial motion ( R 1 ~ 10 7 s −1) persists in the fibrillar state. In the collagen fibril the full width of the glycyl carbonyl powder pattern is 103 p.p.m. This value is substantially smaller than the rigid lattice value, 144 p.p.m., which provides further evidence for motion in the fibril. The observed powder pattern is axially asymmetric, which shows that certain azimuthal orientations are energetically preferred in the fibril. Taken together, the n.m.r. data provide strong evidence that rapid reorientation of the helix backbone occurs in the fibrils. This result shows that formation of a fibrillar structure does not require the existence of a unique set of intermolecular interactions at the helical surfaces.