No abstract

Water hampers the formation of strong and durable bonds between adhesive polymers and solid surfaces, in turn hindering the development of adhesives for biomedical and marine applications. Inspired by mussel adhesion, a mussel foot protein homologue (mfp3S-pep) is designed, whose primary sequence is designed to mimic the pI, polyampholyte, and hydrophobic characteristics of the native protein. Noticeably, native protein and synthetic peptide exhibit similar abilities to self-coacervate at given pH and ionic strength. 3,4-dihydroxy-l-phenylalanine (Dopa) proves necessary for irreversible peptide adsorption to both TiO2 (anatase) and hydroxyapatite (HAP) surfaces, as confirmed by quartz crystal microbalance measurements, with the coacervate showing superior adsorption. The adsorption of Dopa-containing peptides is investigated by attenuated total reflection infrared spectroscopy, revealing initially bidentate coordinative bonds on TiO2, followed by H-bonded and eventually long-ranged electrostatic and Van der Waals interactions. On HAP, mfp3s-pep-3Dopa adsorption occurs predominantly via H-bond and outer-sphere complexes of the catechol groups. Importantly, only the Dopa-bearing compounds are able to remove interfacial water from the target surfaces, with the coacervate achieving the highest water displacement arising from its superior wetting properties. These findings provide an impetus for developing coacervated Dopa-functionalized peptides/polymers adhesive formulations for a variety of applications on wet polar surfaces.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) is involved in both intracellular membraneless organelles and extracellular tissues. Despite growing understanding of LLPS, molecular-level mechanisms behind this process are still not fully established. Here, we use histidine-rich squid beak proteins (HBPs) as model IDPs to shed light on molecular interactions governing LLPS. We show that LLPS of HBPs is mediated though specific modular repeats. The morphology of separated phases (liquid-like versus hydrogels) correlates with the repeats’ hydrophobicity. Solution-state NMR indicates that LLPS is a multistep process initiated by deprotonation of histidine residues, followed by transient hydrogen bonding with tyrosine, and eventually by hydrophobic interactions. The microdroplets are stabilized by aromatic clustering of tyrosine residues exhibiting restricted molecular mobility in the nano-to-microsecond timescale according to solid-state NMR experiments. Our findings provide guidelines to rationally design pH-responsive peptides with LLPS ability for various applications, including bioinspired protocells and smart drug-delivery systems.

Liquid–liquid phase separation (LLPS) of proteins into concentrated microdroplets (also called coacervation) is a phenomenon that is increasingly recognized to occur in many biological processes, both inside and outside the cell. While it has been established that LLPS can be described as a spinodal decomposition leading to demixing of an initially homogeneous protein solution, little is known about the assembly pathways by which soluble proteins aggregate into dense microdroplets. Using recent developments in techniques enabling the observation of matter suspended in liquid by transmission electron microscopy, we observed how a model intrinsically disordered protein phase-separates in liquid environment. Our observations reveal the dynamic mechanisms by which soluble proteins self-organize into condensed microdroplets with nanoscale and millisecond space and time resolution, respectively. With this method, the nucleation and initial growth steps of LLPS could be captured, opening the door for a deeper understanding of biomacromolecular complexes exhibiting LLPS ability.

The beak of the Humboldt squid is a biocomposite material made solely of organic components - chitin and proteins - which exhibits 200-fold stiffness and hardness gradients from the soft base to the exceptionally hard tip (rostrum). The outstanding mechanical properties of the squid beak are achieved via controlled hydration and impregnation of the chitin-based scaffold by protein coacervates. Molecular-based understanding of these proteins is essential to mimic the natural beak material. Here, we present detailed studies of two histidine-rich beak proteins (HBP-1 and -2) that play central roles during beak bio-fabrication. We show that both proteins have the ability to self-coacervate, which is governed intrinsically by the sequence modularity of their C-terminus and extrinsically by pH and ionic strength. We demonstrate that HBPs possess dynamic structures in solution and achieve maximum folding in the coacervate state, and propose that their self-coacervation is driven by hydrophobic interactions following charge neutralization through salt-screening. Finally, we show that subtle differences in the modular repeats of HBPs result in significant changes in the rheological response of the coacervates. This knowledge may be exploited to design self-coacervating polypeptides for a wide range of engineering and biomedical applications, for example bio-inspired composite materials, smart hydrogels and adhesives, and biomedical implants.