Direct observation of structure and dynamics during phase separation of an elastomeric protein - PubMed (original) (raw)
Direct observation of structure and dynamics during phase separation of an elastomeric protein
Sean E Reichheld et al. Proc Natl Acad Sci U S A. 2017.
Abstract
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 cross-linking. 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 ELP3 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.
Keywords: NMR; dynamics; elastin; phase separation; protein structure.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
The intrinsically disordered ELP3 recapitulates the phase behavior and elasticity of tropoelastin materials. (A) CD spectra of ELP3 and tropoelastin monomers in 50 mM sodium phosphate, pH 7 at 20 °C, contain features that are indicative of highly disordered proteins. (B) Bright-field microscopy of temperature-induced phase separation of ELP3 and tropoelastin in 1.5 M NaCl solution after 1 h of incubation at 50 °C. (White scale bars, 32 μm.) (C) Mechanical properties of polymeric ELP3 and tropoelastin coacervates cross-linked at lysine residues by genipin. Error bars correspond to the SEM.
Fig. 2.
The unique dynamics of ELP3 monomers and coacervate facilitate NMR spectroscopy of the phase transition. (A) The 1H-15N HSQC NMR spectrum of 4.7 mM ELP3 monomer in 50 mM sodium phosphate, 300 mM NaCl, pH 7 at 37 °C. Labeled peaks, corresponding to the amino acids in the repeating sequence motifs of the HDs and CLDs, were assigned as described in Materials and Methods and SI Appendix, Fig. S1. To remove temperature effects on chemical shift and protein dynamics, phase separation was induced at 37 °C by increasing the concentrations of both ELP and NaCl. (B) HSQC spectrum of a 7.8-mM ELP3 sample undergoing liquid–liquid phase separation in a buffer consisting of 50 mM sodium phosphate, 600 mM NaCl, pH 7 at 37 °C. (C) HSQC spectra of the same phase-separated sample shown in B, acquired using an R2 relaxation rate filter to remove signals with R2 > 5 s−1 (red) or a PFG diffusion filter (removing objects with a diffusion rate faster than 10−7 cm2⋅s−1) (blue) to distinguish residual monomeric ELP3 (red) from coacervate (blue).
Fig. 3.
The 1HN chemical shift data reflect altered solution conditions within ELP3 coacervates. The HD peaks from 1H-15N HSQC spectra of ELP3 in 50 mM sodium phosphate, pH 7 with NaCl ranging from 0 to 900 mM are shown in each panel, highlighting the correlation between 1HN chemical shifts and the concentration of NaCl. (A) HSQCs acquired at 37 °C with an ELP3 in 50 mM sodium phosphate, pH 7 and 600 mM NaCl undergoing a phase transition are overlaid in black. (B) HSQCs acquired at 20 °C with residual monomer (coacervate was removed by centrifugation) postcoacervation overlaid in black. Changes in ELP3 monomer 1HN chemical shifts are plotted as a function of NaCl concentration at (C) 37 °C or (D) 20 °C, showing a clear linear dependence of chemical shift on salt concentration. Most HDs also exhibited a small protein concentration-dependent change in chemical shift, as shown by the lower than expected Δcs observed in the 0.6 M NaCl sample with 100 mg/mL ELP3. Linear least squares regression of Δcs (red lines) for the site with the smallest protein concentration dependence (G4) was used to estimate the NaCl concentration experienced by residual monomer (red star) or coacervate (red diamond) after LLPS.
Fig. 4.
The intrinsic disorder of monomeric ELP3 is retained after coacervation. (A) Secondary structure propensities from δ2D analysis of NMR chemical shifts. The random coil propensities for each position in the CLDs (i) and HDs (ii) are shown, as are the α-helix propensities for each position in the CLD sequence (iii) and the β-sheet propensities for HD repeat sequences (iv). (B) Schematic representation of the two β-turns identified in the ELP3 NMR data. The colored dashed lines represent the characteristic connectivities between 1H atoms that were used as identifiers of each turn in NOESY spectra of ELP3. (C) Summary of HN-Hα NOEs observed in the HD sequence of the ELP monomer at 20 °C. The bar thickness in each case represents the relative intensity of the NOE cross-peak, with the colored bars corresponding to the dashed lines in A.
Fig. 5.
Intermolecular hydrophobic contacts are formed in the phase-separated state of ELP3. The aliphatic regions of 13C double half filtered 1H-1H NOESY spectra, acquired at 37 °C, are shown for phase-separated (A) and monomeric (B) ELP3. For each sample, spectra show NOEs between 1H-13C groups [double 13C selected (i)]; between 1H-12C and 1H-13C groups [13C selected and filtered (ii); 13C filtered and selected (iii)]; or between 1H-12C groups [double 13C filtered (iv)]. Expansions showing NOEs between Val Hγ and Val Hβ, Pro Hγ, or Ala Hβ are shown in C. For the monomeric sample, only intramolecular contacts (i and iv) within Val side chains are observed in C, and some additional weak intraresidue NOEs are observed (B, iv). In the coacervate, intense NOEs are observed in all spectra, indicating the presence of intermolecular Val–Val, Val–Pro, and Val–Ala contacts. The slight increase in coacervate Val–Val NOEs observed in i and iv is due to the additional presence of intraresidue NOEs in these spectra, as seen for the monomeric ELP3.
Fig. 6.
NMR relaxation and PFG diffusion measurements reveal temperature- and time-sensitive dynamics of phase-separated ELP3. (A) Temperature dependence of ELP3 PFG diffusion rates (i and ii) and 15N R2 or R1 relaxation rates (iii_–_vi). Odd-numbered panels describe the extreme differences in both relaxation properties and translational diffusion between monomeric ELP3 and ELP3 within a coacervate. Even-numbered panels display the relatively subtle differences in monomer ELP3 dynamics caused by variations in protein or salt concentration. Least squared linear regression fits (solid or dashed lines) of the monomer at high and low protein concentrations in the absence of NaCl show the effect of viscosity on these measurements (ii, iv, and vi). The phase transition temperature for a 4.7-mM sample of ELP3 is indicated in each plot by a vertical dashed line. Error bars indicate relaxation rate fitting error or SEM for diffusion measurements. (B) Time-dependent changes in the diffusion and R2 relaxation rates (mean of G1, V2, G4, and G5 data) of the monomeric and coacervate species in an 8.7-mM sample of ELP3 held at 37 °C (phase transition temperature is 30 °C) for 6 d. Error bars indicate the SEM.
Fig. 7.
Model for ELP self-assembly. Our findings provide support for a comprehensive model for ELP self-assembly in which elevated temperature and salt increase the entropic cost of solvating hydrophobic chains, driving phase separation. Because the protein sequence precludes adoption of a stable folded state, the resulting phase-separated state is maintained through transient and nonspecific HD interactions between monomers. Whereas the coacervate remains hydrated, bulk water is excluded and there is a reduction in Na+ and Cl− ions (red dots), slightly increasing their concentration in the bulk solution. The coacervated ELP can be chemically cross-linked to form an elastic material. HDs remain dynamic and highly disordered throughout the assembly process, whereas CLD motion slows with each step. Partially helical in the monomers (blue cylinders), CLDs exhibit an increased propensity to form β-sheets (green arrows) in the coacervate and become stable α-helices in cross-linked materials.
Comment in
- Elastin phase separation - structure or disorder?
Fawzi NL. Fawzi NL. Nat Rev Mol Cell Biol. 2020 Oct;21(10):568-569. doi: 10.1038/s41580-020-00291-0. Nat Rev Mol Cell Biol. 2020. PMID: 32860012 No abstract available.
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