The molecular structuring of complex architectures and the enclosure of space are essential requirements for technical and living systems. Self‐assembly of supramolecular structures with desired ...shape, size, and stability remains challenging since it requires precise regulation of physicochemical and conformational properties of the components. Here a general platform for controlled self‐assembly of tailored amphiphilic elastin‐like proteins into desired supramolecular protein assemblies ranging from spherical coacervates over molecularly defined twisted fibers to stable unilamellar vesicles is introduced. The described assembly protocols efficiently yield protein membrane–based compartments (PMBC) with adjustable size, stability, and net surface charge. PMBCs demonstrate membrane fusion and phase separation behavior and are able to encapsulate structurally and chemically diverse cargo molecules ranging from small molecules to naturally folded proteins. The ability to engineer tailored supramolecular architectures with defined fusion behavior, tunable properties, and encapsulated cargo paves the road for novel drug delivery systems, the design of artificial cells, and confined catalytic nanofactories.
A platform of environmentally controlled architectures ranging from twisted fibers to stable vesicles is demonstrated. Supramolecular architectures are designed via a library of amphiphilic elastin‐like proteins (ELPs). Tailored ELPs, responsive to environmental parameters enable the assembly of complex materials with tunable properties. The described assembly protocols yield Protein Membrane‐Based Compartments enabling the encapsulation of delicate cargo.
The investigation of complex biological processes in vivo often requires defined multiple bioconjugation and positioning of functional entities on 3D structures. Prominent examples include spatially ...defined protein complexes in nature, facilitating efficient biocatalysis of multistep reactions. Mimicking natural strategies, synthetic scaffolds should comprise bioorthogonal conjugation reactions and allow for absolute stoichiometric quantification as well as facile scalability through scaffold reproduction. Existing in vivo scaffolding strategies often lack covalent conjugations on geometrically confined scaffolds or precise quantitative characterization. Addressing these shortcomings, we present a bioorthogonal dual conjugation platform based on genetically encoded artificial compartments in vivo, comprising two distinct genetically encoded covalent conjugation reactions and their precise stoichiometric quantification. The SpyTag/SpyCatcher (ST/SC) bioconjugation and the controllable strain-promoted azide−alkyne cycloaddition (SPAAC) were implemented on self-assembled protein membrane-based compartments (PMBCs). The SPAAC reaction yield was quantified to be 23% ± 3% and a ST/SC surface conjugation yield of 82% ± 9% was observed, while verifying the compatibility of both chemical reactions as well as enhanced proteolytic stability. Using tandem mass spectrometry, absolute concentrations of the proteinaceous reactants were calculated to be 0.11 ± 0.05 attomol/cell for PMBC surface-tethered mCherry-ST-His and 0.22 ± 0.09 attomol/cell for PMBC-constituting pAzF-SC-E20F20-His. The established in vivo conjugation platform enables quantifiable protein–protein interaction studies on geometrically defined scaffolds and paves the road to investigate effects of scaffold-tethering on enzyme activity.
