Biomolecular condensates formed by designer minimalistic peptides

Abstract

Inspired by the role of intracellular liquid-liquid phase separation (LLPS) in formation of membraneless organelles, there is great interest in developing dynamic compartments formed by LLPS of intrinsically disordered proteins (IDPs) or short peptides. However, the molecular mechanisms underlying the formation of biomolecular condensates have not been fully elucidated, rendering on-demand design of synthetic condensates with tailored physico-chemical functionalities a significant challenge. To address this need, here we design a library of LLPS-promoting peptide building blocks composed of various assembly domains. We show that the LLPS propensity, dynamics, and encapsulation efficiency of compartments can be tuned by changes to the peptide composition. Specifically, with the aid of Raman and NMR spectroscopy, we show that interactions between arginine and aromatic amino acids underlie droplet formation, and that both intra- and intermolecular interactions dictate droplet dynamics. The resulting sequence-structure-function correlation could support the future development of compartments for a variety of applications.

Fig. 1. Designer minimalistic peptide droplets.

a Chemical structure of WGR-1. Aromatic amino acid side chains (Trp and Tyr) are colored in blue, Arg side chain is colored in turquoise, and non-polar amino acid side chains that are part of the ELP domain (Pro-Gly-Val-Gly) are colored in orange. b Suggested mechanism of the peptide liquid droplet formation and subsequent partitioning of fluorescent payloads. c Expected intermolecular interactions underlying LLPS of WGR-1 into liquid droplets, including (from left to right) Trp-Trp, Trp-Tyr, Arg-Trp, and Arg-Tyr π-π stacking. Chemical structures of side chains are presented, color coded as described in (a).

Fig. 2. Peptide sequence controls LLPS propensity and droplet formation.

a Table of the designed peptide sequences. b Phase diagram of the peptides as a function of peptide concentration and pH, in tris buffer with 0.2 M NaCl, at room temperature. LLPS was not observed for WGR-2 and WGK. Data are presented as mean values of n = 3 +/− SD. Source data are provided as a Source Data file. c Confocal microscopy bright field images of peptide liquid droplets formed at a concentration of 20 mM in Tris buffer at pH 8 with 0.2 M NaCl. Scale bars = 20 μm. Inset: macroscopic images of the peptide solutions. ‘Source data are provided as a Source Data file.

Fig. 3. Strength of intermolecular interactions affects peptide droplet dynamics.

a–c FRAP analysis of WGR-1, WGR-3, WGR-4, and WGR-5, performed using laser scanning confocal microscopy at 20 mM in Tris buffer at pH 8 with 0.2 M NaCl using 0.5% FITC-labeled peptides. a Representative confocal microscopy images of FRAP for individual droplets. Scale bars = 5 μm. FRAP recovery plots (b), apparent diffusion coefficient (c), and t_1/2 of the recovery (d). Data are presented as mean values +/− SD, n = 5 (WGR-1), 4 (WGR-3, WGR-4), and 5 (WGR-5). Source data are provided as a Source Data file.

Fig. 4. Partitioning of fluorescent payloads within droplets depends on peptide polarity.

Confocal microscopy images of (a) fluorescein, (b) Rhodamine B and (c) GFP partitioning within WGR-1, WGR-3, WGR-4, and WGR-5 peptide droplets. Scale bar = 10 μm. Insets: Macroscopic images of the partitioning of the fluorescent payload in peptide droplets samples before (left) and after (right) centrifugation and droplet sedimentation. Encapsulation efficiency (EE) analysis of peptide droplets calculated from absorbance measurements of bulk solutions for (d) WGR-1 (e) WGR-3 (f) WGR-4 (g) WGR-5. Data are presented as mean of n = 3 +/− SD. Source data are provided as a Source Data file.

Fig. 5. Molecular level analysis of droplet formation by using Raman and NMR spectroscopy.

a Raman spectrum obtained from averaging solution Raman map. b Normalized Raman spectra taken from 3 different spots of the 2D false color image: droplet center (black), droplet edge (red) and the droplet surrounding (light grey). The large peak at 1100 cm⁻¹ originates from the glass background. c False color 3D image showing whole peptide droplet and slice through the center. d False color 2D image created using the 758 cm⁻¹ peak. . One-dimensional ¹³C spectrum of WGR-1 peptide with (red) or without (black) 100 mM NaCl, at 5 mM peptide (non-LLPS, lower spectra) and 20 mM peptide (upper spectra) in 50 mM tris buffer pH 8 and 300 K, chemical shifts are assigned. Inset: averaged Δδ in ¹³C spectra of the four samples for each amino acid at peptide concentration of 5 mM (left bar chart) and 20 mM (right bar chart). Gly were excluded from the analysis due to spectral overlap, and all three Arg were averaged due to partial spectral overlap. Data are presented as mean values +/− SD, n = 8 (W1), 8 (R), 11 (20 mM W9), 10 (5 mM W9), 5 (20 mM P), 4 (5 mM P), 4 (V), 7 (Y). Source data are provided as a Source Data file.

Fig. 6. NMR determines the molecular mechanism of droplet formation.

a The one-dimensional ¹³C spectrum for the WGR-1 peptide at 20 (black, LLPS) and 5 (red, non-LLPS) mM in 50 mM tris buffer pH 10 and 300 K. Arrows indicate chemical shift differences at the Y aromatic ring. b Aromatic region of the 2D-¹H,¹³C-HMBC spectrum showing long-range proton-carbon correlations allowing the detection of quaternary carbons. Y¹⁴ chemical shift changes are shown as before. c Region of the 2D-¹H, ¹H-COSY spectrum showing the correlation between arginine H^γ–H^δ protons.