Mechanistic Insights into the Role of Water in Backbone Dynamics of Native Collagen Protein by Natural Abundance 15N NMR Spectroscopy

Collagen being the most abundant animal protein is an integral component of muscle, connective tissues, cartilage, and bones, having function to provide strength, load bearing, and flexibility to organisms. The structural and functional role of water mediated hydrogen bonding network of collagen has been debated. We present here a solid state nuclear magnetic resonance (ssNMR) spectroscopy method to observe water dependent backbone dynamics of collagen protein in its absolute native environment inside bone. The load bearing function of collagen is directly dependent on its backbone dynamics. The picoseconds backbone dynamics has been measured by relaxation rate measurement (R-1) of natural isotopic abundance N-15 (similar to 0.37%) resonance. The sensitivity of natural abundance N-15 ssNMR spectrum is enhanced by paramagnetic doping of copper-ethylenediaminetetraacetic acid (Cu (II) EDTA) inside bone matrix. Exclusive backbone resonances originating from proline/hydroxyproline (Pro/Hyp) and glycine (Gly) resonances are well resolved in N-15 spectrum, giving access to relaxation rates from native collagen backbone. The water dependent site-resolved relaxation measurements have provided mechanistic insights into role of water mediated hydrogen bonding network in native collagen backbone dynamics. Our study has given new insights in the understanding of water dependent native collagen dynamics, functions, and stability.