Investigations of lysine and lysine-derived crosslinks in elastin via solid-state NMR spectroscopy

Abstract

Elastin is responsible for elasticity and resiliency of force-bearing tissues in vertebrates. This polymeric protein is produced from a ~70 kDa precursor, tropoelastin, During the post-translational modification, a network of inter- and intramolecular covalent crosslinks is formed from lysine residues. As many as eleven different bi-, tri-, and tetrafunctional modifications are present in mature elastin, which are critical for normal tissue function. These products have only been characterized in hydrolysates and enzymatically-cleaved peptides, but never observed directly in the intact protein. Two isotopes of lysine, [U-13C,15N-Lys] and [13Cε-Lys], were independently incorporated into the neonatal rat smooth muscle cell (NRSMC) elastin. Samples with high levels of enrichment (~90%) were used for high-resolution solid-state nuclear magnetic resonance (ssNMR) studies. One- and two-dimensional measurements allowed for analysis of the secondary structures of lysines. Random coil and α-helices were the predominant conformations. The helical content is higher in a frozen sample than at the physiological temperature. Experiments based on cross-polarization allowed for quantification of unmodified lysine as well as desmosine and isodesmosine, crosslinks unique to elastin. The presence of rare bi- and trifunctional crosslinks was confirmed.