A Two-State Model Describes the Temperature-Dependent Conformational Equilibrium in the Alanine-Rich Domains in Elastin.

Elastin is the insoluble elastomeric protein that provides extensibility and resilience to vertebrate tissues. Limited high-resolution structural data for elastin are notably complex. To access this information, this protein is considered in the simplified context of its two general domain types, that is, hydrophobic (HP) and crosslinking (CL). The question of elastin's structure-function has directed the focus of nearly all previous studies in the literature to the unique repeating sequences characteristic of this protein, found primarily in the HP domains. The CL domains were assumed to play a very limited role in biological elasticity due in part to the significant α-helical character that was (incorrectly) predicted for these regions. In this study, the conformational heterogeneity of alanines in native elastin's CL domains is examined in the context of helix-coil transition theory (HCTT) using solid-state nuclear magnetic resonance (SSNMR) spectroscopy in tandem with strategic isotopic labeling. Helix and coil populations are observed at all temperatures, but the former increases significantly at lower temperatures. Below the glass transition temperature (Tg), two major populations of alanines in the CL regions are resolved by two-dimensional SSNMR; one-dimensional methods are used for characterization in nativelike conditions. The spectra of 13CO-Ala in the CL regions are simulated using an HCTT-based statistical mechanical representation. Below Tg, longer segments with significant helical probabilities are consistent with the experimental data. At higher temperatures, the SSNMR lineshapes are best fit with a distribution of shorter (Ala)n segments, most in random coil. These results are used to refine a structure-function model for elastin in the context of HCTT, redirecting attention to the CL domains and their role in elasticity.