Keeley, F. W. | Search bioRxiv

bioRxiv2026

A free energy landscape screen reveals the disordered conformational ensemble of tropoelastin

Understanding how proteins explore their conformational energy landscapes is essential for linking sequence to function, yet current ensemble methods are limited by sampling inefficiency and poor scalability to large disordered systems. Here we introduce a free energy landscape screen (FELS), a conceptually different approach that replaces sampling-centric ensemble fitting with broad exploration of energy landscapes, screening thousands of landscape shapes--from highly funneled to flat and rugged. By systematically biasing and evaluating large conformer pools according to contact propensities derived from experiment, FELS efficiently identifies sets of conformers that best reproduce experimental data and highlights candidates for structural refinement, without being restricted by chain length or amount of disorder. To demonstrate the power of this approach we applied it to a previously intractable system, human tropoelastin (hTE), a [~]700-residue precursor of elastin. FELS provides the first experimentally defined atomistic view of the hTE conformational ensemble, revealing that this protein is intrinsically disordered yet exhibits distinct local secondary structure and specific, transient medium- and long-range contacts that organize its ensemble. These findings reconcile long-standing conflicting models and demonstrate that FELS provides a general, experimentally driven framework for mapping conformational energy landscapes of large proteins across the continuum between structural order and disorder.

biochemistry↗

bioRxiv2024

Evolutionary Constraints on Positional Sequence, Collective Properties and Sequence Style of Tropoelastin Dictated by Fundamental Requirements for Formation and Function of the Extracellular Elastic Matrix

Elastin is an unusual extracellular matrix protein responsible for the properties of extension and energy-efficient elastic recoil of large blood vessels, heart valves, lung parenchyma and many other vertebrate tissues requiring such resilience. Polymeric elastin is assembled from monomeric tropoelastin by a process involving liquid-liquid phase separation, followed by maturation into an extended elastic matrix, covalently cross-linked through the side chains of lysine residues in the protein and producing a robust biomaterial with the structural integrity to withstand hundreds of millions of cycles of extension and recoil without mechanical failure. Elastin functions as an entropic elastomer, whose properties are the direct result of the inability of the protein to fold into a fixed, stable structure. Previous investigations of how the unusual properties of polymeric elastin arise from the sequence of tropoelastin have depended primarily on modeling using molecular biological and biophysical methodologies. This study takes a unique alternative approach, using a well-curated database of tropoelastin sequences from more than 80 representative species of Amniotes to identify characteristics that are conserved over more than 300 million years of evolution in order to provide assembly and conformational flexibility requirements of elastins. Conserved characteristics included preservation not only of regions of linear or positional sequence, but also of collective or compositional characteristics, derived from the sequence but not strictly dependent on positional sequence. A plausible overall consensus sequence for Amniote tropoelastins allowed quantification of residue-by-residue, domain-by-domain and region-by-region levels of sequence conservation, identifying distinct regions of high and low positional sequence conservation. Regions of low positional sequence conservation nevertheless maintained a recognizable, low complexity sequence style characterized by tandem repeats and partial repeats of short, non-polar motifs. Analysis of these motifs indicated hPGhGG, with numerous mutations, insertions and deletions, as the underlying repeating unit in all Amniote tropoelastins. Together these data identify significant evolutionary constraints dictated by fundamental requirements for formation and functionality of the extracellular elastin matrix. Mutations/polymorphisms in human tropoelastin affecting such well-conserved characteristics might be expected to have phenotypic consequences.

biochemistry↗