Investigations of the Unique Role of Alanines in the 'Elastin Puzzle' by Solid-State Nmr Spectroscopy and Molecular Dynamics Simulations

Abstract

Elastin is the main protein component of the elastic fiber that provides extensibility and resilience to vertebrate tissues. Despite its importance to tissue functionality, elastin’s high-resolution molecular structure remains elusive. Structural determination using solution-state nuclear magnetic resonance (NMR) spectroscopy or X-ray diffraction is not applicable to this polymer, due to its insolubility and non-crystallinity. Elastin is the mature crosslinked aggregates of tropoelastin, a ~70 kDa soluble monomeric protein consisting of alternating hydrophobic and crosslinking domains. Tropoelastin’s crosslinking domains are characterized by two or three lysines in alanine-rich sequences, such as AAAAKAAKAA and AAAKAAAKAA; the hydrophobic regions are identified by tandem repeats of nonpolar amino acids such as PGVGVA. The abundance of alanines in the two domain types provides an opportunity to probe structural and dynamical microenvironments throughout the polymer. Hence, the characterization of alanines in this protein serves as a significant step to solving the “elastin puzzle”. New insights into the structure and dynamics of elastin are provided by solid-state NMR (ssNMR) spectroscopy and molecular dynamics (MD) simulations. Elastin samples with high levels of isotopic enrichment (~80%) at the alanines were expressed by neonatal rat smooth muscle cells (NRSMC) culture. The results of one- and two-dimensional ssNMR experiments indicate a heterogeneity of conformation and dynamics of alanines in this crosslinked polymer, elastin. Variable-temperature ssNMR and MD studies suggest a conformational equilibrium between α-helix and random coil populations in the crosslinking domains. The application of helix-coil transition theory to the interpretation of ssNMR lineshapes provides new insights to understanding elastin’s molecular structure. The Ala-rich crosslinking domains undergo rapid helix-coil interconversions, whereas the hydrophobic domains are characterized by random coil, which exhibits fast large-amplitude motions. Based on these results, an updated model is proposed to describe elastin’s molecular organization, reflecting the dynamic characteristics of this protein.