Research ArticleMATERIALS SCIENCE

Intrinsically disordered proteins access a range of hysteretic phase separation behaviors

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Science Advances  18 Oct 2019:
Vol. 5, no. 10, eaax5177
DOI: 10.1126/sciadv.aax5177
  • Fig. 1 LCST IDPPs exhibit a wide range of hysteretic phase behaviors.

    (A) Analysis of the reversible phase behavior of LCST IDPPs in our library revealed three groups of repeat motifs, wherein motifs in each group encode one of three types of phase behavior characterized by differences in the degree of thermal hysteresis seen on cooling below the cloud point temperature, ranging from (i) negligible (~0°C) and (ii) moderate (10° to 30°C) to (iii) large, environmentally sensitive hysteresis. Here, we show temperature-dependent optical turbidity over a full cycle of heating and cooling pass the Tcp for three representative IDPPs that exhibit the full range of observed hysteretic behaviors. As a guide to the eye, each panel includes a legend with a qualitative indicator of the degree of hysteresis for each repeat motif. (B) IDPPs made of (VAPVG) repeats exhibit highly reproducible degrees of thermal hysteresis over multiple cycles of phase separation. (C) Extension of data in (A) examining the phase behavior of (VGAPVG)35 to show its large, environmentally sensitive hysteresis, as it shows (in separate experiments) large or negligible thermal hysteresis depending on the maximum temperature (shown by arrows) reached during the heating part of the cycle. (D) Hysteretic phase behavior of IDPPs with an increasing number of (VAPVG) repeats. (E) Analysis of IDPPs in (D) but varying the cooling rate (from 1° to 0.1°C/min). To improve data visualization, the corresponding Tcp on heating are shown as vertical dashed lines. All optical turbidity measurements were performed at a fixed concentration of 50 μM in PBS, with heating and cooling at 1°C/min, unless otherwise stated.

  • Fig. 2 Hysteretic LCST IDPPs within the marked hysteretic regime exhibit structural dynamics that deviate from the behavior of canonical IDPs.

    (A and B) CD data as a function of temperature for two IDPPs, consisting of either 35 (A) or 80 (B) (VAPVG) repeats and displaying ~5°C wide (A) or ~15°C wide (B) thermal hysteresis in their phase behavior when triggered at an IDPP concentration of 5 μM in water. (C and D) CD data as a function of temperature for two IDPPs, consisting of either 30 (C) or 80 (D) (TPVAVG) repeats. Solutions of both IDPPs at a concentration of 5 μM in water display large degrees of thermal hysteresis (>50°C) in their phase behavior. For all IDPPs shown here, the Tcp was calculated from optical turbidity data obtained under the same experimental conditions (5 μM in water) as our CD spectroscopy data. For simplicity, mean residue ellipticity (θ) values are shown as θ*10−3.

  • Fig. 3 A protein denaturant modulates the hysteretic phase behavior of LCST IDPPs.

    (A) Cloud point temperatures as a function of increasing concentrations of a protein denaturant (urea) in PBS for IDPPs with negligible—(VPGVG)40 and (VPGVG)80; see fig. S5—moderate—(VAPVG)40 and (VAPVG)80—and large—(TPVAVG)40, (TPVAVG)80, (VGPVG)40, and (VGPVG)80—hysteresis. Values were normalized to Tcp in PBS without urea. To overcome the overlapping of normalized Tcp’s, these data are also presented as separate panels in fig. S5. (B) Degree of thermal hysteresis for IDPPs made of VAPVG repeats at a fixed concentration of 50 μM in PBS supplemented with increasing amounts of urea. (C) Temperature-dependent optical turbidity data in PBS + 1 M urea for two IDPPs with identical repeat number but different repeat motifs that encode widely different hysteretic behaviors. Even in 4 M urea, 40-mer IDPPs of TPVAVG repeats display pronounced hysteretic behavior (fig. S5). (D) Phase behavior data as in (C), in PBS with 1 or 2 M urea, but exclusively for (TPVAVG)80 and when the phase transition is triggered without exceeding 45°C during the heating cycle. All relevant Tcp values were calculated from optical turbidity data at a concentration of 50 μM in PBS or PBS supplemented with urea as indicated.

  • Fig. 4 Subtle changes in repeat syntax can have profound effects on the hysteretic phase behavior of IDPPs and their related native IDPs.

    (A) Peptide motifs that relate to each other by simple sequence reversal—rewriting a peptide sequence by reading it from C to N terminus—present identical patterns of amino acid side chains, which we illustrate with the structure of a pentapeptide motif and its reversed motif as observed in the crystal structures of two different proteins (Protein Data Bank ID 3MKR_B and 1OZP, respectively). The images were rendered using PyMOL (http://pymol.org/). (B to E) Temperature-dependent optical turbidity to probe the phase behavior of four IDPP pairs, wherein each pair consists of two IDPPs with identical number of repeats of motif sequences that are interrelated by sequence reversal. As a guide to the eye, each panel includes a legend with a qualitative indicator of the degree of hysteresis for each IDPP. Figure S7 shows CD spectroscopy at 25°C that reaffirms the intrinsic disorder of these IDPP pairs before their phase transition. (F) Pro and Gly content of a representative group of Pro- and Gly-rich IDPs that we previously characterized as being enriched in Pro-Xn-Gly motifs (10). (G) Enrichment or depletion of Gly in residue positions surrounding P-Xn-G motifs among Pro- and Gly-rich IDPs, expressed as a fold change from the random occurrence of Gly based on total Gly content. Asterisks indicate significant (P < 0.001) divergence from a random distribution (see Supplementary Methods). (H) Temperature-dependent turbidimetry of an IDPP composed of a motif wherein Gly occurs one residue N-terminal to P-Xn-G and corresponding mutant polymers wherein Gly was substituted by bulkier amino acids. All turbidity measurements were conducted in PBS at an IDPP concentration of 50 μM, except for VRPVG (+1 M NaCl).

  • Fig. 5 MD simulations of syntactically related IDPPs reveal interchain interaction forces that promote hysteresis.

    (A and B) Simulations of 18-mer, single IDPP chains at low (290 to 310 K) and high (350 to 390 K) temperatures for three IDPP pairs studied in Fig. 4 (C to E). (A) Fraction of residues that are part of unstructured motifs for single IDPP chains at high and low temperatures (see fig. S9 for all other structural motifs). (B) Temperature-dependent changes in radius of gyrations (Rg, black) and absolute peptide-water interaction energy (Epw, blue), expressed as a differential between values at high and low temperatures (ΔRg and ΔEpw). Dashed lines between data points for IDPPs in each pair are guides to the eye. (C and D) Simulations of two closely interacting 18-mer IDPP chains for each IDPP in Fig. 4 (C to E) to study interchain interactions in a model phase separated state. (C) Fraction of value changes (f) in interchain interaction quantities (see Supplementary Methods for details) after cooling these two chain “phase separated” systems to 290 K for 25 ns. f = (end value − initial value)/(initial value). (D) Snapshots from our two-chain simulations for an IDPP with marked hysteresis (VGAPVG)18 (Fig. 4D) and for an IDPP with negligible hysteresis (LGAPVG)18 (Fig. 4E). The production simulations were performed for 25 ns with a 2-fs time step.

  • Fig. 6 Hysteretic LCST phase behavior enables the synthesis of morphologically diverse nanoparticles that resist disassembly.

    (A) The nonequilibrium phase behavior of hysteretic LCST IDPPs may affect the thermally triggered assembly of protein-based block copolymers into micelles. (B and C) By synthesizing diblock copolymers composed of a common hydrophilic, corona-forming ELP block (VPGXG)80, where X = [A:G], and hydrophobic, core-forming blocks that repeat a hysteretic LCST motif with either (B) moderate (VAPVG) or (C) large (TPVAVG) hysteresis, we synthesized self-assembling nanoparticles that remain assembled below the critical assembly temperature in proportion to the degree of thermal hysteresis of the core-forming block. Their temperature-dependent assembly was studied using UV-visible spectroscopy, shown as small changes in optical turbidity (<0.4 U at 350 nm, circles), and by dynamic light scattering that revealed large changes in hydrodynamic radius (Rh, diamonds). Heating and cooling were performed at 1°C/min. Error bars are the measured polydispersity. (D and E) Cryo-TEM images show that diblock IDPPs with similar block architecture and hydropathy balance assemble into distinct rod-like morphologies by virtue of the unique hysteretic phase behavior of the core-forming blocks. Scale bars in large fields of view are 200 nm. Scale bars within insets are 50 nm. (F) Time-dependent stability of nanoparticles formed by a diblock copolymer with the same repeat composition and block ratio as in (C) and (E) but having half the number of repeats in the (TPVAVG) core. Stability was studied as normalized optical turbidity (normalized to time zero) as a function of time at two constant temperatures (arrows) that are well below the critical assembly temperature—inset shows temperature-dependent changes in optical turbidity (O.T.).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax5177/DC1

    Supplementary Methods

    Table S1. DNA sequence information for synthesis of genes encoding IDPPs by Pre-RDL.

    Fig. S1. LCST IDPPs display large, environmentally sensitive hysteresis.

    Fig. S2. Repeat number influences the hysteretic phase behavior of LCST IDPPs.

    Fig. S3. Forms of irreversible phase behavior in UCST IDPPs.

    Fig. S4. LCST IDPPs exhibit CD spectra characteristic of intrinsic disorder and regardless of their hysteretic nature.

    Fig. S5. Effect of urea on the hysteretic phase behavior of IDPPs.

    Fig. S6. Imaging of nonhysteretic and hysteretic IDPPs upon phase separation.

    Fig. S7. Secondary structure of IDPPs related by sequence reversal at the repeat level.

    Fig. S8. Steric hindrance at the residue position preceding a P-Xn-G motif influences hysteresis.

    Fig. S9. Secondary structure preferences calculated from single-chain IDPP simulations at low and high temperatures.

    References (4453)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • Table S1. DNA sequence information for synthesis of genes encoding IDPPs by Pre-RDL.
    • Fig. S1. LCST IDPPs display large, environmentally sensitive hysteresis.
    • Fig. S2. Repeat number influences the hysteretic phase behavior of LCST IDPPs.
    • Fig. S3. Forms of irreversible phase behavior in UCST IDPPs.
    • Fig. S4. LCST IDPPs exhibit CD spectra characteristic of intrinsic disorder and regardless of their hysteretic nature.
    • Fig. S5. Effect of urea on the hysteretic phase behavior of IDPPs.
    • Fig. S6. Imaging of nonhysteretic and hysteretic IDPPs upon phase separation.
    • Fig. S7. Secondary structure of IDPPs related by sequence reversal at the repeat level.
    • Fig. S8. Steric hindrance at the residue position preceding a P-Xn-G motif influences hysteresis.
    • Fig. S9. Secondary structure preferences calculated from single-chain IDPP simulations at low and high temperatures.
    • References (4453)

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