Targeting of proteins and protein analogs to metal-chelating lipid vesicles

Citation

Shnek, Deborah Rebecca (1996) Targeting of proteins and protein analogs to metal-chelating lipid vesicles. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ctnn-4057. https://resolver.caltech.edu/CaltechETD:etd-01032008-092017

Abstract

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This investigation demonstrates that new metal-chelating lipids formed into mixed vesicles can bind to a variety histidine-rich ligands through metal coordination. Our results show the feasibility of metal-chelating lipids as a method for targeting histidine-rich compounds to lipid interfaces. Interesting metal chelating lipid materials for protein orientation studies, matching the surface distribution of surface residues, protein drug delivery, and for two-dimensional protein crystallization could be made with chelating-lipids. Engineered proteins containing a histidine "tag" or proteins with natural surface histidine residues are easily targeted to the interface using metal binding.

Both metal binding and histidine-rich ligand binding were investigated with mixed metal-chelating lipid vesicles. New metal-chelating lipids containing an iminodiacetate (IDA) chelating-moiety were formed into mixed vesicles and shown to bind transition metal ions through the lipid headgroup. Metal binding was characterized through calorimetry, freeze-etch microscopy, light scattering, ESR, and fluorescence studies. Fluorescently-labeled lipids containing iminodiacetate showed a large change in the fluorescence emission spectra upon metal binding, behavior which has proven useful for a vesicle-based metal sensor.

Metal-loaded vesicles bound a model protein specifically through surface-accessible histidines, as shown using ESR studies. Equilibrium binding measurements showed at least an order of magnitude increase in binding affinity of the protein for the membrane when metal was present. The association constants determined through isothermal titration calorimetry for a model bivalent histidine compound binding to metal-chelating lipid bilayers were of the order of [...], while monovalent binding constants were of the order [...].

Lipid re-organization upon ligand binding was probed with model histidine compounds and histidine polymers using fluorescently-labeled metal-chelating lipid vesicles. The ability of lipids to form multivalent ligand-lipid complexes was investigated using the formation of lipid excimers, as demonstrated by fluorescence measurements of the E/M intensity ratio. Histidine content of model compounds determines the magnitude of the effect on the fluorescence emission spectra. Histidine polymers showed a larger increase of the E/M ratio than the smaller bivalent or monovalent compounds. Combining equilibrium binding results obtained with model complexes and results from fluorescence experiments, studies with metal-chelating vesicles support multivalent coordination and reorganization of the lipids by histidine-rich ligands.

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