Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity

Abstract

Elastin enables the reversible deformation of elastic tissues and can withstand decades of repetitive forces. Tropoelastin is the soluble precursor to elastin, the main elastic protein found in mammals. Little is known of the shape and mechanism of assembly of tropoelastin as its unique composition and propensity to self-associate has hampered structural studies. In this study, we solve the nanostructure of full-length and corresponding overlapping fragments of tropoelastin using small angle X-ray and neutron scattering, allowing us to identify discrete regions of the molecule. Tropoelastin is an asymmetric coil, with a protruding foot that encompasses the C-terminal cell interaction motif. We show that individual tropoelastin molecules are highly extensible yet elastic without hysteresis to perform as highly efficient molecular nanosprings. Our findings shed light on how biology uses this single protein to build durable elastic structures that allow for cell attachment to an appended foot. We present a unique model for head-to-tail assembly which allows for the propagation of the molecule's asymmetric coil through a stacked spring design.

Fig. 1.

Diagrammatic representation of all the tropoelastin constructs used in this study. Black boxes represent hydrophilic domains and white boxes represent hydrophobic domains. The dotted line indicates the absence of exon 26A which is rarely present in tropoelastin.

Fig. 2.

Small angle X-ray and neutron scattering of full-length human tropoelastin. (A, i) The experimental SAXS (squares) and SANS (triangles) data are plotted as a function of q, and compared with a typical theoretical fit obtained with GASBOR (solid line). (ii) The low angle regions of the X-ray scattering data were analyzed in the form of Guinier plots (log I vs. q²), from which the radius of gyration (Rg) can be extracted from the slope (Rg²/3) of the straight line. The slope demonstrates the expected linearity for the values q ≤ 1/Rg (shaded region). (iii) Pair distribution function calculated for the SAXS (squares) and SANS (triangles) datasets. The curves show with error bars the distribution of interatomic spacings, with maxima at 22 nm (SAXS) and 20 nm (SANS). (iv) Kratky plots are shown for the SAXS (squares) and SANS (triangles) data. (B, i) GASBOR ab initio shape of full-length tropoelastin calculated from solution SAXS data, 20 models are shown superimposed. (ii) Filtered average shape of 20 individual SAXS (blue) and SANS (gray) simulations. (iii) Superimposed filtered average SAXS and SANS models. (iv) Labeled diagram of the model for full-length tropoelastin showing proposed locations of the N terminus, the spur region containing exons 20–24, and the C terminus. (Scale bar, 5 nm).

Fig. 3.

Small angle X-ray scattering of overlapping fragments of human tropoelastin. Ab initio models were calculated from SAXS data for tropoelastin constructs 2–18 (pink), 2–25 (green), and full-length tropoelastin (blue) and from SANS data for full-length tropoelastin (gray). The models are shown both individually and superimposed in three orthogonal views.

Fig. 4.

Characterization of the mechanical properties of single-tropoelastin molecules. (A) Example of a force-extension curve for a single molecule. The red line corresponds to a fit to the worm-like-chain model of polymer elasticity using 211 nm for contour length and 0.38 nm for the persistence length. (C) Superposition of several (n = 17) force-extension curves for the stretching of tropoelastin molecules. (B, D) Frequency histograms for persistence lengths, p, and contour lengths, ΔL_c. The mean values of persistence and contour lengths are 0.36 ± 0.14 nm (n = 158) and 166 ± 49 nm (n = 158), respectively.

Fig. 5.

(A) This plot shows a typical experiment where a molecule was extended (blue trace) and relaxed (red trace) two times and then it was overextended in order to detach it from the surface (black trace). This last trace has a single detachment force peak consistent with the stretching pattern of a single molecule. (B) The forward and reverse traces are well characterized by the WLC equation (dotted line) and there is no hysteresis in the time scale of the experiment (∼500 ms). (C) Histogram for the persistence length shows a mean of 0.35 ± 0.09 nm (n = 147; 26 molecules).

Fig. 6.

Head-to-tail model for nascent elastic fiber assembly. (A) Juxtaposed domains 19 and 25 on one tropoelastin molecule and domain 10 on an adjacent monomer. (B) Molecular schematic of the cross link interface. (C) Tandem assembly displaying n-mer propagation as an outcome of covalently bonded molecules.