Lipidation alters the phase-separation of resilin-like polypeptides

Abstract

Biology exploits biomacromolecular phase separation to form condensates, known as membraneless organelles. Despite significant advancements in deciphering sequence determinants for phase separation, modulating these features in vivo remains challenging. A promising approach inspired by biology is to use post-translational modifications (PTMs)-to modulate the amino acid physicochemistry instead of altering protein sequences-to control the formation and characteristics of condensates. However, despite the identification of more than 300 types of PTMs, the detailed understanding of how they influence the formation and material properties of protein condensates remains incomplete. In this study, we investigated how modification with myristoyl lipid alters the formation and characteristics of the resilin-like polypeptide (RLP) condensates, a prototypical disordered protein with upper critical solution temperature (UCST) phase behaviour. Using turbidimetry, dynamic light scattering, confocal and electron microscopy, we demonstrated that lipidation-in synergy with the sequence of the lipidation site-significantly influences RLPs' thermodynamic propensity for phase separation and their condensate properties. Molecular simulations suggested these effects result from an expanded hydrophobic region created by the interaction between the lipid and lipidation site rather than changes in peptide rigidity. These findings emphasize the role of "sequence context" in modifying the properties of PTMs, suggesting that variations in lipidation sequences could be strategically used to fine-tune the effect of these motifs. Our study advances understanding of lipidation's impact on UCST phase behaviour, relevant to proteins critical in biological processes and diseases, and opens avenues for designing lipidated resilins for biomedical applications like heat-mediated drug elution.

Fig. 1. Schematic phase diagram and architecture of proteins in this study. (a) A homogeneous solution of RLP in buffer (1 − Φ) undergoes spontaneous phase separation upon cooling below its phase boundaries, forming a suspension of two immiscible phases (2 − Φ): protein-rich condensates in a protein-poor solvent. (b) Disordered RLP sequence in purple, with two distinct lipidation sites, differing by three amino acids (in blue and red). Each lipidation site can be modified with a myristoyl group (grey).

Fig. 2. Characterization of UCST phase behaviour and concentration-dependence of cloud temperatures for unmodified and lipidated RLPs. (a) Representative variable-temperature turbidimetry plots for AGA-RLP (dashed line) and m-AGA-RLP (solid line) at 20 μM in PBS. Both constructs exhibited UCST behaviour, with a sharp increase in solution turbidity once the temperature was reduced below their respective cloud temperature (T_c). (b) Partial temperature-composition phase diagrams, illustrating the dilute branch of the phase diagram. Lipidation enhanced the phase separation propensity of RLPs, but the extent of this effect depended on the sequence of the lipidation sites. Error bars are standard deviations from two independent measurements, while the dashed line represents the 95% confidence interval of the linear regression.

Fig. 3. Characterization of temperature-dependent phase separation and condensate morphology using dynamic light scattering and fluorescent microscopy. (a) Temperature-dependent changes in the hydrodynamic radius of proteins monitored by DLS. Unmodified constructs exhibited sharp aggregation from unimers below their T_c. Lipidated constructs show self-assembly at temperatures higher than T_c, with sizes dependent on the lipidation site sequence. [protein] = 10 μM in PBS, with error bars representing standard deviations from three measurements. (b) and (c) Visualization of the condensates of lipidated constructs using confocal microscopy at 25 °C. [protein] = 100 μM in PBS. See Fig. S10 (ESI†) for non-lipidated samples. m-AGA-RLP formed spherical condensates, whereas m-LSL-RLP formed clusters of smaller droplets.

Fig. 4. Characterization of condensate fluidity using fluorescent recovery after photobleaching (FRAP). (a) Output frames from confocal microscopy are presented at various time points: t = −3 s before bleaching, immediately after bleaching (t = 0 s), and at subsequent intervals post-bleaching to observe recovery. (b) and (c) The FRAP data are normalized and analysed using a single exponential model (see methods for details) to determine the half-life of fluorescence recovery. Two-way ANOVA indicates a statistically significant interaction between the effects of lipidation and lipidation site sequence (F(1, 8) = 18.47, p = 0.0030). In the absence of lipidation, the recovery is rapid and does not depend on the sequence of the lipidation site (Tukey's HSD, p > 0.9999). Lipidation, however, slows the rate of recovery, and the recovery rate of m-LSL-RLP is significantly slower than that of m-AGA-RLP (Tukey's HSD, p = 0.0013).

Fig. 5. Nano-scale organization of lipidated RLPs condensates. Cryo-TEM and negative-stain TEM micrographs of m-AGA-RLP (a), (c) and (e) and m-LSL-RLP (b), (d) and (f) reveal the influence of lipidation sites on the morphology of protein chains within the condensates. m-AGA-RLP forms diffuse, shadow-like structures, whereas m-LSL-RLP shows clusters of aggregate. An 'x' marks an ice contamination on the cryo-TEM grid in panel c.

Fig. 6. Computational analysis of lipidation site hydrophobicity using the PARCH scale method. This analysis revealed distinct differences in PARCH values between lipidated RLPs, with m-LSL-RLP displaying higher hydrophobicity (denoted by lower PARCH values) compared to m-AGA-RLP, even in regions where both constructs share similar amino acid sequences. These findings suggest that the interplay between the lipidation site and lipid may lead to an extended hydrophobic region, likely enhancing the interactions between lipidated RLPs in solution and condensed phases.