Deuterium nuclear magnetic resonance of specifically labeled native collagen. Investigation of protein molecular dynamics using the quadrupolar echo technique

Abstract

Collagen was labeled with [3,3,3-d3]alanine and with [d10]leucine via tissue culture. 2H nuclear magnetic resonance (NMR) spectra were obtained of collagen in solution and as fibrils using the quadrupolar echo technique. The 2H NMR data for [3,3,3-d3]alanine-labeled collagen fibrils were analyzed in terms of a model for motion in which the molecule is considered to jump between two sites, separated azimuthally by an angle 2 delta, in a time which is rapid compared with the residence time in both sites. The data suggest that the molecule undergoes reorientation over an angle, 2 delta, of approximately 30 degrees in the fibrils, and that the average angle between the alanine C alpha--C beta bond axis and the long axis of the helix is approximately 75 degrees. Reorientation is possibly segmental. The T2 for [3,3,3-d3]alanine-labeled collagen fibrils was estimated to be 105 mus. The 2H NMR data for the methyl groups of [d10]leucine-labeled collagen were analyzed qualitatively. These data established that for collagen in solution and as fibrils, rotation occurs about the leucine side-chain bonds, in addition to threefold methyl rotation and reorientation of the peptide backbone. The T2 for the methyl groups of leucine-labeled collagen is estimated to be approximately 130 mus. Taken together, these data provide strong evidence that both polypeptide backbone reorientation and amino acid side-chain motion occur in collagen molecules in the fibrils. Stabilizing interactions that determine fibril structure must therefore depend upon at least two sets of contacts in any given local region.

Characterization of leucine side-chain reorientation in collagen-fibrils by solid-state 2H NMR.

“…Sci. USA 79 (1982) (,Ir I shown that many sets of local contacts exist between collagen molecules (1)(2)(3)(4)(5). The present results suggest a definite model for the conformational changes ofthe leucine side chain that take place within the mobile contact region between collagen molecules.…”

“…300 (1)(2)(3)(4). These studies also have provided strong evidence that the amino acid side chains exhibit extensive internal motions in the fibrils (3,5 (9) that accounts for spectral distortion due to a finite pulse width. Lorentzian line broadening was used.…”

A current viewpoint on structure and evolution of collagens. I. Fibrillar collagens

“…Even in mineralized tissues the collagen fibrils represent dynamic structures. The neighbor monomer molecules are established to be oriented azimuthly in fibril and to perform rotation near their longitudinal axis [54]. Such fluctuations of monomers also promote acceleration of formation of contacts between telopeptides and spiralizes sites of neighbor tropocolagen molecules and hence of the collagen molecule self-organization and the fibril growth.…”

The Molecular Organization of Polymers of Cartilage in Health and Disease

“…There were also reports where deuterium NMR was used to describe the molecular motions of collagen within the cartilage [108,109]. Although these experiments were very helpful, they were extremely expensive since labelling of selected amino acids of collagen was required [108].…”

“…Although these experiments were very helpful, they were extremely expensive since labelling of selected amino acids of collagen was required [108]. The most important result of these studies was that the cartilage collagen remains essentially rigid as opposed to the highly mobile GAGs.…”