Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy

Ohgo, Kosuke; Niemczura, Walter P.; Seacat, Brian C.; Wise, Steven G.; Weiss, Anthony S.; Kumashiro, Kristin K.

doi:10.1074/jbc.m111.285163

Cited by 18 publications

(40 citation statements)

References 46 publications

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“…This evidence for fluctuating β-turns is consistent with spectroscopic data ( Muiznieks et al, 2010 ; Tamburro et al, 2003 ) but not with the β-spiral, a highly-ordered structural model of elastin which requires that repeated β-turns be formed simultaneously, ( Venkatachalam and Urry, 1981 ) which is never observed in the simulations. As such, our results reconcile spectroscopic evidence for local secondary structure ( Muiznieks et al, 2010 ; Tamburro et al, 2003 ; Ohgo et al, 2012 ) with global conformational disorder.…”

Section: Resultssupporting

confidence: 81%

The liquid structure of elastin

Rauscher

Pomès

2017

eLife

165

163

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The protein elastin imparts extensibility, elastic recoil, and resilience to tissues including arterial walls, skin, lung alveoli, and the uterus. Elastin and elastin-like peptides are hydrophobic, disordered, and undergo liquid-liquid phase separation upon self-assembly. Despite extensive study, the structure of elastin remains controversial. We use molecular dynamics simulations on a massive scale to elucidate the structural ensemble of aggregated elastin-like peptides. Consistent with the entropic nature of elastic recoil, the aggregated state is stabilized by the hydrophobic effect. However, self-assembly does not entail formation of a hydrophobic core. The polypeptide backbone forms transient, sparse hydrogen-bonded turns and remains significantly hydrated even as self-assembly triples the extent of non-polar side chain contacts. Individual chains in the assembly approach a maximally-disordered, melt-like state which may be called the liquid state of proteins. These findings resolve long-standing controversies regarding elastin structure and function and afford insight into the phase separation of disordered proteins.

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Section: Resultssupporting

confidence: 81%

The liquid structure of elastin

Rauscher

Pomès

2017

eLife

165

163

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“…Solid-state NMR has been used previously to provide information on the structure of insoluble elastin (9,(77)(78)(79), but these investigations have generally focused on characterization of the mobile hydrophobic domains of the protein and provided little or no insight into detailed structure of the cross-linking domains. Furthermore, no previous NMR study has addressed the question of the structure of elastin in the coacervated state.…”

Section: Discussionmentioning

confidence: 99%

Conformational Transitions of the Cross-linking Domains of Elastin during Self-assembly

Reichheld

Muiznieks

Stahl

et al. 2014

Journal of Biological Chemistry

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Background: Elastin is a polymeric protein providing extensibility and elastic recoil to tissues. Results: Cross-linking domain structure shifts from random coil to ␤-strand to ␣-helix during assembly of elastin matrix. Conclusion: Cross-linking domains have a previously unappreciated structural lability during assembly, which is highly susceptible to mutations of lysine residues. Significance: Identification of conformational transitions in cross-linking domains of elastin during self-assembly is essential for understanding the mechanisms of formation of the elastic matrix.

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“…In contrast, the CLDs provide structural integrity to polymeric elastin through covalent cross-linking of lysine residues (27,28). Current knowledge of elastin structure comes primarily from low-resolution spectroscopic and solid-state NMR studies (29)(30)(31)(32)(33)(34)(35),…”

mentioning

confidence: 99%

Direct observation of structure and dynamics during phase separation of an elastomeric protein

Reichheld

Muiznieks

Keeley

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

225

245

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Despite its growing importance in biology and in biomaterials development, liquid-liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical crosslinking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient β-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP 3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.phase separation | elastin | NMR | protein structure | dynamics T he liquid-liquid phase separation (LLPS) of molecules has long been exploited to concentrate and encapsulate molecules for drug delivery and food preparation (1, 2). In biology, there is increasing awareness that many proteins exhibit this type of phase behavior, allowing transient microenvironments to be quickly assembled and disassembled in response to changing solution conditions, or the availability of binding partners (3, 4). Protein phase separation occurs intracellularly to generate various membraneless organelles, such as ribonucleoprotein (RNP) bodies involved in nucleic acid processing, transport, and storage (5). A similar phenomenon is observed in the extracellular matrix as a critical step in the synthesis of elastic fibers, which provide extensibility, recoil, and resilience to tissues (6, 7). In the latter system, LLPS of monomeric elastin results in hydrated protein-rich coacervate droplets that are deposited and crosslinked to form an elastic matrix (7,8). In addition to their fundamental importance to biology, the dynamic reversibility of LLPS makes phase-separated states of proteins an attractive platform for development of responsive biomaterials with broad application, for instance as scaffolds for tissue engineering or as carriers in drug delivery systems (9-11).Despite the keen interest in proteins that undergo s...

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Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy

Cited by 18 publications

References 46 publications

The liquid structure of elastin

The liquid structure of elastin

Conformational Transitions of the Cross-linking Domains of Elastin during Self-assembly

Direct observation of structure and dynamics during phase separation of an elastomeric protein

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