Alzheimer's disease, among other neurologically degenerative diseases, has been linked to protein-enzyme interactions that originate within the transmembrane domain of a cell. The lipid environment that houses these interactions lends difficulty to studying intramembrane interactions, often making for time consuming data analysis. Lengthy data interpretation on top of the rate in which protein-enzyme interactions take place creates a need for a method to overcome these obstacles. The use of lipid vesicle fusion to apply a margin of control over the time frame of interaction combined with deep probing spectroscopic techniques can minimize the interference of the lipid environment. Deep ultraviolent resonance Raman (dUVRR) is a vibrational spectroscopic technique that probes along the protein backbone that allows for removal of lipid environmental interference through background subtraction. Lipid vesicle fusion has been demonstrated by mixing lipid vesicles comprised of oppositely charged head groups (cationic 1,2-diaurroyl-sn-glycero-3-phospho-(1-rac-glycerol) (DLPG)) and anionic 1,2-dilauroyl-sn-glycero-3-ethylphocholine (12:0 EPC) or 1,2-dimyristoyl-sn-glycero-3-ethylphocholine (14:0 EPC)) of equivalent or varying aliphatic tail length, up to a 2-carbon difference. The fluorescent dye, 8-aminonapthalene 1,3,6-trisulfonic acid (ANTS), paired with the quencher, p-xylene-bis-pyridiumbromide (DPX), are separately encased in either DLPG or 12:0 EPC/14:0 EPC, respectively, in aqueous solution, and evidence of lipid vesicle fusion is provided by monitoring fluorescence intensity of ANTS as the two solutions are mixed, resulting in the closing proximity of ANTS and DPX observed as a decrease in fluorescence intensity. Additional evidence is provided by dynamic light scattering (DLS) measurements of both independent vesicle solutions and their mixture showing an increase in hydrodynamic radius (Rh). In addition, cohesion of similarly sized lipids is demonstrated, as DLPG (12-carbon chain) fails to fuse with cationic lipids of chain length 16 carbons or longer. Circular dichroism (CD) is a spectroscopic technique that uses left and right-handed polarized light to obtain the overall secondary structure of proteins. Shown is the use of CD to probe the secondary structure and the changes incurred on PolyLA7 (PLA7), a model ?-helical peptide when placed in a transmembrane or hydrophobic environment, through a change in the lipid environment. PLA7 was inserted in DLPG lipid vesicles and then mixed in solution with lipid vesicles comprised of 14:0 EPC. CD spectra were obtained pre and post vesicle fusion, demonstrating the use of lipid fusion as a means to combine membrane embedded proteins of interest while still being able to observe changes that take place. Finally, we propose an on-demand lipid fusion system in which two separate lipid vesicles could be co-suspended in solution and then chemically or photonically induced to fuse. A titration was performed to obtain the pKa of a synthesized pH inducible cationic lipid (pHiCL). The pHiCL is a dipicolylamine with an attached 12-carbon aliphatic tail. The pHiCL was titrated while suspended in an aqueous environment and while inserted into a lipid vesicle comprised of DLPC, a net neutral lipid also with a 12-carbon length aliphatic tail. The pHiCL will be the first component of a two-part system in which a photoacid (PA) will be used to protonate the pHiCL in solution giving rise to cationic and anionic surfaced lipid vesicles causing vesicle fusion to occur.
A nano flow cytometer for single lipid vesicle analysis
