Tuning the Structural Integrity and Mechanical Properties of Globular Protein Vesicles by Blending Crosslinkable and NonCrosslinkable Building Blocks

Abstract

Vesicles made from functionally folded, globular proteins that perform specific biological activities, such as catalysis, sensing, or therapeutics, show potential applications as artificial cells, microbioreactors, or protein drug delivery vehicles. The mechanical properties of vesicle membranes, including the elastic modulus and hardness, play a critical role in dictating the stability and shape transformation of the vesicles under external stimuli triggers. Herein, we have developed a strategy to tune the mechanical properties and integrity of globular protein vesicle (GPV) membranes of which building molecules are recombinant fusion protein complexes: a mCherry fused with an acidic leucine zipper (mCherry-Z E ) and a basic leucine zipper fused with an elastin-like polypeptide (Z R -ELP). To control the mechanical properties of GPVs, we introduced a nonstandard amino acid (paraazidophenylalanine (pAzF)) into the ELP domains (ELP-X), which enabled the creation of crosslinked vesicles under ultraviolet (UV) irradiation. Crosslinked GPVs made from mCherry-Z E /Z R -ELP-X complexes presented higher stability than noncrosslinked GPVs under hypotonic osmotic stress. The degree of swelling of GPVs increased as less crosslinking was achieved in the vesicle membranes, which resulted in the disassembly of GPVs into membraneless coacervates. Nanoindentation by atomic force microscopy (AFM) confirmed that the stiffness and Young's elastic modulus of GPVs increase as the blending molar ratio of Z R -ELP-X to Z R -ELP increases to make vesicles. The results obtained in this study suggest a rational design to make GPVs with tunable mechanical properties for target applications by simply varying the blending ratio of Z R -ELP and Z R -ELP-X in the vesicle selfassembly.

Tuning C-Phycocyanin Photoactivity via pH-Mediated Assembly–Disassembly

“…SDS-PAGE analysis suggested that C-PC was also covalently modified by UV irradiation in a pH-dependent manner (see Figure S11A for details). Generally, photo-crosslinking in proteins often occurs between several photoactive residues, such as Cys, His, Trp, and Tyr, forming disulfide, histidine–lysine, or dityrosine linkages. − In our case, the photo-crosslinkingoccurred intermolecularly via nonreducible covalent bondsmost likely involves Tyr residues, given their high abundance in C-PC relative to the other photoactive residues (Figure S3A). Moreover, it was noticed that most of the Tyr residues in C-PC locate around the chromophores and at the interfaces across α- and β-subunits (Figure ), making it possible for the photo-crosslinking to happen intermolecularly across separate chains, as was suggested by SDS-PAGE gels.…”

Temperature-responsive membrane permeability of recombinant fusion protein vesicles

“…8,[12][13][14][15] Protein vesicles or proteinosomes, consisting of fully folded, functional globular proteins, have recently attracted attention due to their expected biological properties, including biocompatibility and specific protein activities in vesicle structure, useful for microbioreactors, drug delivery vehicles, and synthetic protocells. 3,[16][17][18][19] Particularly, globular protein vesicles (GPVs) can be made through self-assembly of recombinant fusion proteins consisting of fully folded, globular proteins and intrinsically disordered polypeptides in benign aqueous environments at room temperature. 16,17 Since this fabrication is favorable to biological molecules and processes, GPVs can offer diverse biochemical functionality and tunability.…”

Protein Vesicles with pH-Responsive Disassembly

“…34 Due to the behavior of ELPs, self-assembly of vesicles is dependent on ionic strength, protein concentration, temperature, and composition of the ELP guest residue. 29,34,36 Vesicle self-assembly also depends on the characteristics of the globular protein due to the molecular packing constraints of the amphiphiles including the molar ratio of globule-Z E to Z R -ELP (Z E /Z R ratio). 31,32 Here, we incorporated the ionizable amino acid histidine into the guest residue of ELP to develop pH-sensitive vesicles.…”

“…The vesicles are composed of a single layer with a hydrophilic shell of globular protein on the exterior and hydrophobic ELP on the interior . Due to the behavior of ELPs, self-assembly of vesicles is dependent on ionic strength, protein concentration, temperature, and composition of the ELP guest residue. ,, Vesicle self-assembly also depends on the characteristics of the globular protein due to the molecular packing constraints of the amphiphiles including the molar ratio of globule-Z E to Z R -ELP (Z E /Z R ratio). , …”