Multiscale simulations enable thermodynamic and structural characterization of ELP condensates. (A) Illustration of the sequence for the simulated diblock ELPs that consist of five-amino acid repeats, where X is substituted with a guest amino acid. (B) Overview of the three-step multiscale simulation approach that gradually increases the model resolution. Simulations of V5L5 were used to produce the example configurations at each step. Peptides are shaded red-white, while water molecules, chlorine, and sodium, are colored in blue, green, and orange, respectively. Inserts of a G-V-G repeat and surrounding water molecules are shown to indicate the resolution of each model. (C) Correlation between the simulated surface tension (τ) of 20 ELP condensates and the transition temperatures (Tt) of related systems.46 ρ is the Pearson correlation coefficient between the two data sets, and the dashed line is the best fit between simulation and experimental data. Error bars represent the standard deviation of estimates from five independent time windows.

Internal organization of ELP condensates for (A) V5F5, (B) V5L5, (C) V5A5, and (D) V5G5. The uppermost panels present representative configurations from MARTINI simulations for each system. Periodic images along the x and y dimensions are shown for clarity, and the condensate-water interface is perpendicular to the z-axis. Only proteins are shown, with the X- and V-substituted halves of the peptides shown in red and blue, respectively. The central panels show contact maps between amino acids from different peptides. The blue and red bars indicate the V- and X-substituted half of the peptides. The lower panels plot the radial distribution functions, g(r), for amino acids only from the V-substituted half of the peptides (V5-V5), only from the X-substituted half of the peptides (X5-X5), and between the two halves (V5-X5). We limited the calculations to amino acid pairs from different peptides.

Experimental support of the microphase separation of ELP condensates. (A) Reversible ELP condensates formation via changing temperature. NHS ester fluorophores are attached at the amino-termini of V30X30 and X30V30 to detect the different micro-physicochemical properties between the V-end and the X-end. (B) Structures of NHSBODIPY and NHS-SBD, and FLIM images of V30A30, A30V30, V30G30 and G30V30 labeled with respective fluorophores. The fluorescence lifetime of each image is the average acquired from three independent experiments. Scale bar: 5 µm. (C) Schematic diagram of optical tweezers stretching experiment. (D) Bright field images of V30A30 and A30V30 in the stretching experiment using the optical tweezer. Scale bar: 2 µm. (E) Normalized droplets fluorescence intensity changes while stretching (red curve: V30A30; blue curve: A30V30). Fifteen droplets (at size ∼ 4µm) were imaged and used for statistical analysis.

Interior of ELP condensates exhibits interfacial properties as a result of microphase separation. (A) Pearson correlation coefficient, ρ, between the stated measure of hydrophobicity and the condensate transition temperature, Tt, measured by Urry.46 To remove discrepencies in which end of the scale is hydrophobic and which end is hydrophilic, all hydrophobicity scales, including the Urry scale, are first normalized such that 1 corresponds to the most hydrophobic residue and 0 corresponds to the least hydrophobic residue. Hydrophobicity measures considered include experimental measures of water-solvent transfer free energies (water-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine[POPC]-interface,76 water-octanol1,77 water-octanol2,78 water-ethanol,78,79 water-cyclohexane,80 and water-N-cyclohexyl-2-pyrrolidone[CHP]81), atomic level analysis of moieties within amino acids (Kapcha),83 computational approaches to estimate hydrophobicity within disordered proteins (IDPs184 and IDPs285), bioinformatics techniques that approximate the burial propensity, or relative solvent accessibility (RSA) of amino acids (RSA186 and RSA287), and a method that mixed protein burial fraction with water-vapor transfer free energy (RSA/watervapor88). The red box highlights the high correlation between Tt and water-octanol or water-POPC-interface transfer free energies. (B, C) Condensate organization from MARTINI simulations for V5F5 (B) and V5A5 (C). The top panels provide protein-only views for simulated condensates, with the guest residue X, glycine adjacent to the guest residue, and the remaining residues colored red, green, and blue, respectively. Condensate images are repeated periodically in the x/y plane. The bottom panel shows the overall radial distribution function from the guest amino acid to those amino acids native to the ELP sequence.

Water hydrogen bonding environment of condensates from all-atom simulations. (A) Bar chart depicting the average number of protein-water (PW, in green) and protein-protein (PP, in red) hydrogen bond (HB) per residue for each condensate system. The x-axis presents the guest residue of the system and is ordered by the Urry scale. Error bars represent standard deviations of four independent estimates. (B) Water HB density for each of the 20 condensates, for three different residue selections. The metric is shown for all residues in the system (blue), guest residues (purple), and glycine residues adjacent to the guest residue in the sequence (orange). The x-axis presents the guest residue of the system and is ordered by the Urry scale. Error bars represent standard deviations of four independent estimates. (C) Correlation coefficients of the water HB density shown in part B with Urry Tt. (D) Schematics depicting two systems with low (i) and high (ii) water hydrogen bond density. The protein is depicted as blue and orange lines, and the water molecules are depicted as purple circles. Protein-protein and protein-water hydrogen bonds are drawn as red and green dashed lines respectively. (E) Visualizing hydrogen bond density with illustrative snapshots for V5F5 (i) and V5G5 (ii) condensate. Side chain carbon atoms are shown in purple. Water molecules near the protein are explicitly depicted, with oxygen and hydrogen atoms colored in red and white. Water molecules hydrogen bonded to the protein are depicted as spheres (with hydrogen bonds shown in green lines).

Comparison of protein conformation and solvation between ELP condensates and monomers. (A) Zoom in view of a single peptide chain (red) from atomistic simulations of V5A5 condensate (left) and monomer (right). Atoms within one nm of the peptide are shown, including water (cyan), chlorine (green), sodium (orange), and protein atoms from other chains (gray). (B) RSA of guest residues estimated from atomistic simulations of condensates (blue) and monomers (orange). For comparison, the corresponding values estimated using folded proteins are included as purple dots. 87 Error bars represent the standard deviation of four independent time estimates. (C) Comparison of the radius of gyration (Rg) for peptides estimated from atomistic simulations of condensates (blue) and monomers (orange). The dashed line represents the expected Rg of an ideal chain, utilizing values that have been previously suggested for IDPs.91,92 Error bars represent the standard deviation of four independent time estimates.