The
increasing realization of the prevalence of liquid–liquid
phase separation (LLPS) across multiple length scales of biological
constructs, from intracellular membraneless organelles to extracellular
load-bearing tissues, has raised intriguing questions about intermolecular
interactions regulating LLPS at the atomic level. Squid-beak derived
histidine (His)- and tyrosine (Tyr)-rich peptides (HBpeps) have recently emerged as suitable short model peptides to precisely
assess the roles of peptide motifs and single residues on the phase
behavior and material properties of microdroplets obtained by LLPS.
In this study, by systematically introducing single mutations in an
HBpep, we have identified specific sticker residues
that attract peptide chains together. We find that His and Tyr residues
located near the sequence termini drive phase separation, forming
interaction nodes that stabilize microdroplets. Combining quantum
chemistry simulations with NMR studies, we predict atomic-level bond
geometries and uncover inter-residue supramolecular interactions governing
LLPS. These results are subsequently used to propose possible topological
arrangements of the peptide chains, which upon expansion can help
explain the three-dimensional network of microdroplets. The stability
of the proposed topologies carried out through all-atom molecular
dynamics simulations predicts chain topologies that are more likely
to stabilize the microdroplets. Overall, this study provides useful
guidelines for the de novo design of peptide coacervates
with tunable phase behavior and material properties. In addition,
the analysis of nanoscale topologies may pave the way to understand
how client molecules can be trapped within microdroplets, with direct
implications for the encapsulation and controlled release of therapeutics
for drug delivery applications.