In two separate systems, we have demonstrated selective vesicle fusion with biological recognition motifs not natively associated with lipid bilayer fusion, thus broadening the scope of recognition-guided membrane activation. These findings have resonance with goals in targeted chemical delivery and nanoscale compartmentalized chemistry.
Our first system employs two complimentary surface bound fusogens: vancomycin glycopeptide coupled to the antimicrobial peptide magainin II, and D-Ala-D-Ala-OH dipeptide coupled to a phospholipid derivative. Fusion was characterized by dynamic light scattering and FRET experiments with lipid bound fluorophores. We have demonstrated that appropriately designed membrane anchored molecular recognition motifs have the ability to activate specific membrane merging. We further characterized this fusion reaction with regard to the following variables: effects of fusogen concentration, lipid composition, and membrane charge. The results indicate that these parameters are determinants of fusion rate, vesicle stability, peptide binding, catalytic fusion, and membrane disruption during fusion. Notably, these data highlight the importance of coupling between molecular recognition and insertion for bilayer activation as well as the critical role of membrane subdomain formation for membrane fusion reactivity. The phenomena are general to lipid membrane chemistry, and therefore our findings provide a guideline for understanding more complex biomembrane systems.
In our second fusion system, cyanuric acid (CA) and melamine (M) functionalized lipids in membranes exhibited robust hydrogen-bond driven surface recognition in water, facilitated by multivalent surface clustering of recognition groups and variable hydration at the lipid-water interface. We introduced a minimal recognition cluster: three CA or M recognition groups were forced into proximity by covalent attachment to a single lipid headgroup. This trivalent lipid system guides recognition at the lipid-water interface using cyanurate-melamine hydrogen bonding when incorporated at 0.1-5 mole percent in fluid phospholipid membranes, inducing both vesicle-vesicle binding and membrane fusion. Fusion was accelerated when the antimicrobial peptide magainin was used to anchor trivalent recognition, or when added exogenously to a pre-assembled lipid vesicle complex, underscoring the importance of coupling recognition with membrane disruption in membrane fusion. Membrane apposition and fusion were studied in vesicle suspensions using light scattering, FRET assays for lipid mixing, surface plasmon resonance and cryo-electron microscopy. Recognition was found to be highly spatially selective as judged by vesicular adhesion to surface patterned supported lipid bilayers (SLBs). Fusion to SLBs was also readily observed by fluorescence microscopy. Together, these studies indicate effective and functional recognition of trivalent phospholipids, despite low concentration, and solvent competition for hydrogen bond donor/acceptor sites. This novel designed molecular recognition motif may be useful for directing aqueous-phase assembly and biomolecular interactions.
Lastly, we extend our study to other molecular recognition driven events such as phagocytosis. Preliminary experiments are carried out in regard to the affinity labeling of phosphatidylserine receptors in cell lysates and the design of phosphatidylserine modified polymers as mimics of apoptotic cells. Current results on affinity labeling of PS-receptors suggests possible proteins that are selectively labeled but they need to be further confirmed by mass spectroscopy. While uptake of the first generation of PS-polymers with FITC tags is successful, subsequent cytokine production still needs to be verified, and better controls need to be applied.