Biological membranes play an essential role in living organisms by providing stable and functional compartments, preserving cell architecture, whilst supporting signalling and selective transport that are mediated by a variety of proteins embedded in the membrane. However, mimicking cell membranes - to be applied in artificial systems - is very challenging because of the vast complexity of biological structures. In this respect a highly promising strategy to designing multifunctional hybrid materials/systems is to combine biological molecules with polymer membranes or to design membranes with intrinsic stimuli-responsive properties. Here we present supramolecular polymer assemblies resulting from self-assembly of mostly amphiphilic copolymers either as 3D compartments (polymersomes, PICsomes, peptosomes), or as planar membranes (free-standing films, solid-supported membranes, membrane-mimetic brushes). In a bioinspired strategy, such synthetic assemblies decorated with biomolecules by insertion/encapsulation/attachment, serve for development of multifunctional systems. In addition, when the assemblies are stimuli-responsive, their architecture and properties change in the presence of stimuli, and release a cargo or allow "on demand" a specific in situ reaction. Relevant examples are included for an overview of bioinspired polymer compartments with nanometre sizes and membranes as candidates in applications ranging from drug delivery systems, up to artificial organelles, or active surfaces. Both the advantages of using polymer supramolecular assemblies and their present limitations are included to serve as a basis for future improvements.
Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy.
The development of advanced stimuli-responsive systems for medicine, catalysis, or technology requires compartmentalized reaction spaces with triggered activity. Only very few stimuli-responsive systems preserve the compartment architecture, and none allows a triggered activity in situ. We present here a biomimetic strategy to molecular transmembrane transport by engineering synthetic membranes equipped with channel proteins so that they are stimuli-responsive. Nanoreactors with triggered activity were designed by simultaneously encapsulating an enzyme inside polymer compartments, and inserting protein "gates" in the membrane. The outer membrane protein F (OmpF) porin was chemically modified with a pH-responsive molecular cap to serve as "gate" producing pH-driven molecular flow through the membrane and control the in situ enzymatic activity. This strategy provides complex reaction spaces necessary in "smart" medicine and for biomimetic engineering of artificial cells.
Compartmentalization of functional biological units, cells, and organelles serves as an inspiration for the development of biomimetic materials with unprecedented properties and applications in biosensing and medicine. Because of the complexity of cells, the design of ideal functional materials remains a challenge. An elegant strategy to obtain cell-like compartments as novel materials with biofunctionality is the combination of synthetic micrometer-sized giant unilamellar vesicles (GUVs) with biomolecules because it enables studying the behavior of biomolecules and processes within confined cavities. Here we introduce a functional cell-mimetic compartment formed by insertion of the model biopore bacterial membrane protein OmpF in thick synthetic membranes of an artificial GUV compartment that encloses-as a model-the oxidative enzyme horseradish peroxidase. In this manner, a simple and robust cell mimic is designed: the biopore serves as a gate that allows substrates to enter cavities of the GUVs, where they are converted into products by the encapsulated enzyme and then released in the environments of GUVs. Our bioequipped GUVs facilitate the control of specific catalytic reactions in confined microscale spaces mimicking cell size and architecture and thus provide a straightforward approach serving to obtain deeper insights into biological processes inside cells in real time.
Light-driven proton pumps, such as proteorhodopsin, have been proposed as an energy source in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like proto-or nanocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into these membranes is delicate and quantitative studies comparing these two systems are needed. Here we present a detailed analysis of the formation of proteoliposomes and proteopolymersomes and the requirements for a successful reconstitution of the membrane protein proteorhodopsin. To this end, we apply design of experiments to provide a mathematical framework for the reconstitution process. Mathematical optimization identifies suitable reconstitution conditions for lipid and polymer membranes and the obtained data fits well to the predictions. Altogether, our approach provides experimental and modeling evidence for different reconstitution mechanisms depending on the membrane type which resulted in a surprisingly similar performance.
For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system.
For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a protonpumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/ off depending on the requirements of the molecular system.
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