pH-Responsive Elastin-Like Polypeptide Designer Condensates

Abstract

Biomolecular condensates are macromolecular complexes formed by liquid–liquid phase separation. They regulate key biological functions by reversibly compartmentalizing molecules in cells, in a stimulus-dependent manner. Designing stimuli-responsive synthetic condensates is crucial for engineering compartmentalized synthetic cells that are able to mimic spatiotemporal control over the biochemical reactions. Here, we design and test a family of condensate-forming, pH-responsive elastin-like polypeptides (ELPs) that form condensates above critical pH values ranging between 4 and 7, for temperatures between 20 and at 37 °C. We show that the condensation occurs rapidly, in sharp pH intervals (ΔpH < 0.3). For eventual applications in engineering synthetic cell compartments, we demonstrate that multiple types of pH-responsive ELPs can form mixed condensates inside micron-sized vesicles. When genetically fused with enzymes, receptors, and signaling molecules, these pH-responsive ELPs could be potentially used as pH-switchable functional condensates for spatially controlling biochemistry in engineered synthetic cells.

Understanding the Phase Behavior of a Multistimuli-Responsive Elastin-like Polymer: Insights from Dynamic Light Scattering Analysis

“…We hypothesize that this difference may be attributed to the lower linear charge density of KI8, leading to greater susceptibility to screening effects. We can also compare this result to a recent systematic study of acidic pH-responsive ELPs of varying length and composition . The authors of this study also find that some of their ELPs are pH-insensitive under certain conditions despite having many ionizable residues.…”

“…For example, for an ELP-based biosensor to function in biologically relevant conditions, it should ideally respond to a single stimulus, such as binding to a target biomolecule, while remaining insensitive to variations in local pH, electric potential, ion concentration, and the presence of other cosolutes, at least for the duration and conditions of the experiment. In bulk solution, the stimuli-responsive behavior of ELPs is characterized by a reversible transition between soluble, extended nanoscale polypeptide chains and micron-scale coacervates formed when the hydrophobic regions of the ELP seek burial through intra- and intermolecular association. − This phenomenon, which has been referred to as coacervation, liquid–liquid phase separation (LLPS), aqueous two-phase systems (ATPS), and lower-critical solution temperature (LCST) behavior, has been exploited to create various architectures and assemblies. − When coacervate assembly is not possible or favorable, such as when ELPs are end-tethered polymers to a solid substrate or in single-molecule simulations, single-chain ELPs will still undergo continuous collapse resembling a coil-to-globule transition, accompanied by changes to internal degrees of freedom, hydration, and stiffness. − …”

Contribution of the ELRs to the development of advanced in vitro models

“…ELRs containing charged amino acids in their sequence are responsive to pH changes ( Ribeiro et al, 2009 ; MacKay et al, 2010 ). By controlling the proportion of charged (i.e., Glu and His) and hydrophobic (i.e., Val, Ile, Phe and Tyr) amino acids in the guest position, de Haas et al engineered pH-responsive ELRs with the capability to form condensates under narrow pH changes within a physiologically relevant range (i.e., pH 4–7) ( de Haas et al, 2023 ).…”

Peptide-Mediated Nanocarriers for Targeted Drug Delivery: Developments and Strategies

“…Temperature influences peptide conformation and solubility [ 98 ]. Elastin-like peptides (ELPs) exhibit significant solubility shifts at specific temperatures [ 99 ], which have been applied to various drug delivery systems.…”