Lipid bilayers and embedded membrane proteins that anchor or span the lipidic leaflets are the fundamental molecular assemblies that constitute cellular membranes in all living organisms. By acting as receptors, channels, and transporters, transmembrane proteins allow communication, signaling, and solute translocation across the membrane insulating barrier. For the structural and mechanistic characterization of membrane proteins, the scientific community currently relies on detergents to solubilize and purify membrane protein as protein-detergent micelle complexes that then can be investigated through a plethora of biochemical and biophysical approaches. In addition, to mimic the native lipid bilayer environment and generate a barrier that separates milieus of different solute compositions, membrane proteins can be reconstituted in artificial lipid bilayer vesicles of controlled size, called proteoliposomes, which allow investigating molecular processes like cargo translocation and generation of electrochemical gradients across the bilayer. Although these approaches are desirable, stability issues related to the intrinsic hydrophobic nature of membrane proteins and the lability of interactions between protein, detergent, or lipids within micelles and unilamellar liposomes limit the widespread application of these systems. Since these supramolecular assemblies are metastable in nature, the community is continuously searching for new strategies to stabilize these systems against physical and chemical stressors.
The laboratory of Dr. Gassensmith at the Department of Chemistry and Biochemistry at UT Dallas, together with several other research groups, has pioneered the use of crystalline metal-organic framework (MOFs) scaffolds to embed and stabilize purified biomolecules. On the other end, our laboratory focuses on characterizing the mechanism of substrate transport of various classes of transmembrane metal transporters that catalyze the translocation of essential and toxic transition metals across the membranes. Dr. Gassensmith and I envisioned that we could combine our expertise and develop a strategy towards stabilizing both micellar and liposomal particles.
In one of the many discussions that we had, we realized that the stabilization of supramolecular assemblies as delicate as liposomes, membrane protein-detergent micelles, and proteoliposmes could be achieved via a biomineralization-like process through the formation of crystalline exoskeletons that would act as a shield against stressors.
In the design, an ideal supramolecular scaffold's desired properties included its assembly from simple biologically-compatible building blocks to form structures with high thermodynamic stability yet high kinetic lability that would allow rapid disassembly when required. These properties would allow the exoskeleton to assemble rapidly on the biomolecular complexes, thereby increasing their stability and guarantee that that shell removal could be achieved effectively, allowing for the full recovery of functional biomolecular assemblies.
At first, we sought to develop the biomineralization process to stabilize protein-free liposomal preparations and then systematically expanded the strategy to transmembrane protein systems and proteoliposomes. We selected two transmembrane metal transporters investigated in my laboratory (a primary active Cu(I) P-type ATPase pump, CopA, and a ferrous iron solute carrier-like transporter, IroT) as a proof of principle to showcase the potential generalizability of the approach.
The purified metal transporters utilized in the study are very susceptible to denaturation or inactivation, they typically need to be stored at low temperatures, and the corresponding proteoliposomes need to be generated via extrusion immediately before their analysis. We developed detailed protocols to rapidly assemble a crystalline zeolitic imidazole framework-8 (ZIF-8) scaffold composed of zinc and 2-methyl imidazole (2-MIM) on micelles and proteoliposomes and developed a compatible strategy for shell disassembly by chelation with EDTA (Scheme 1).
Dr. Gassensmith's team, together with our colleague Dr. Smaldone, were able to characterize in detail the crystallinity and structural properties of the formed ZIF-8 exoskeleton. Upon exfoliation of the ZIF shell, we were amazed that we could recover micellar and proteoliposome preparations that were structurally and functionally indistinguishable from pristine freshly prepared samples. In light of the exoskeleton's stability, we postulated that the systems would be resilient to both aging, physical and chemical stressors. Indeed, the approach allowed the encapsulated samples to resist high temperatures, mechanical stresses, exposure to denaturants, and resilience against prolonged aging. Dr. Gassensmith decided to mail encapsulated samples from Texas to Rhode Island and back in standard cushioned mailer. Upon return the samples were stored at room temperature for two months, and upon exfoliation proteoliposome morphology and protein activity were similar to the pristine samples. To gain a detailed understanding of exoskeleton formation, we realized that we needed to dissect the process of biomineralization in detail. We postulated that our approach harness the ability of liposomes and proteoliposomes to template the ZIF-8 growth. Our students (Fabian, Sameera, and Nisansala) successfully demonstrated that the weak interaction between zinc and the negatively charged groups on lipidic polar heads could template the growth of the ZIF-8 exoskeleton directly on the surface of the vesicles, allowing the generated scaffold to thoroughly coat the lipid bilayer assemblies and "freezing" them in the crystalline structure. Dr. Gassensmith went a step further and contacted the team of Dr. Falcaro at the Graz University of Technology in Austria to inquire about the possibility of conducting sophisticated synchrotron-based in-situ SAXS/WAXS measurements to monitor the rapid kinetic of exoskeleton nucleation, particle growth, crystallization, and morphology. With great excitement, the analysis demonstrated, as we hypothesized, that the liposomes induce faster nucleation, growth, and crystallization of ZIF compared to controls.
The developed technology combines attractive features. The chemicals utilized for the exoskeleton assembly are cheap and straightforward (zinc acetate and 2-MIM). The high thermodynamic stability of the ZIF framework guarantees that the exoskeleton can act as a "shield" against stressors by immobilizing the lipid-protein assemblies in a rigid structure. The kinetic labile nature of the coordination bonds in the shell allows for rapid disassembly of the structure by simple EDTA chelation. Upon "exfoliation," all the exoskeleton building blocks can be easily removed with a simple chromatographic step. This guarantees a straightforward procedure for the assembly and disassembly of ZIF, in which Zn(II) and 2-MIM can act as molecular Lego-blocks to build a scaffold around the liposome particles.
The project undoubtedly required and benefited from the combination of our diverse expertise, spanning from membrane protein biochemistry, supramolecular chemistry, and material development and characterization. The collaborative effort proved critical for tackling this intriguing problem, bringing together four research groups from the USA and Europe. This is a beautiful example demonstrating how scientists possessing different skills can synergize to develop new approaches that interface the biomolecular world with the material realm.
We believe that this technology can open new opportunities from basic science to biotechnology and medicine applications. The ability to stabilize and maintain the integrity of liposomes, membrane protein-micelle complexes, and proteoliposomes will facilitate the exchange of materials for basic research across research laboratories in different locations. In light of the use of liposomal preparations for drug delivery and liposomal vaccine development, we can envision that stabilization of membrane protein-lipid bilayer assemblies via immobilization in ZIFs might provide a new tool to potentially overcome the challenges of "cold-chain" distribution of these unstable systems. We have learned about the challenges for the storage and transport of liposomal vaccines during the COVID-19 pandemic, and we hope that our work can contribute to new applications in the biomedical field.