Characterizing the interactions of molecules with phospholipid vesicles and bilayers by confocal raman and single-molecule fluorescence microscopy

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Publication Type dissertation
School or College College of Science
Department Chemistry
Author Myers, Grant Aaron
Title Characterizing the interactions of molecules with phospholipid vesicles and bilayers by confocal raman and single-molecule fluorescence microscopy
Date 2012-12
Description Molecular interactions with phospholipid bilayers are investigated using two types of laser-based microscopies. In optical-trapping confocal Raman microscopy, individual phospholipid vesicles are held in place in situ with laser tweezers while Raman scattering spectra are collected to report the preconcentration of analyte molecules via pH-gradient loading. A theory of accumulation is developed to account for source-depletion and vesicle buffering, factors that can significantly reduce the total accumulation. The system of 10 equations including 10 unknowns, five equilibrium constants and five experimental parameters was solved with symbolic-based mathematics software to yield a sixth-order polynomial having a single physically-meaningful, positive, real root. Utilizing this theory, an experiment is designed in which a single vesicle is manipulated by optical tweezers into isolation from other interfering vesicles and subjected to a variety of solution conditions. Raman spectra are analyzed quantitatively with a classical least squares method to determine the concentrations of compounds inside of the vesicle. When the pH gradient is 6.5 units and the citric-acid buffer inside of vesicles is 0.5 M, a 104-fold preconcentration factor of a model compound benzyl-dimethyl amine is observed, where accumulation into a 600-nm diameter vesicle is complete in less than 20 min. Total-internal-reflection fluorescence (TIRF) microscopy is a technique with the dynamic range, spatial and temporal resolution capable of characterizing the interactions iv of single fluorescently labeled peptides with a planar supported lipid bilayer. TIRF images are analyzed with an adjacent pixel criterion to discriminate between molecular events and random noise exceeding the threshold. The bi-exponential distribution of event durations is interpreted with respect to a consecutive reversible three-state kinetics model, where unfolded peptides from solution first interact weakly with the bilayer then fold to persist for a longer time. Application of this model is shown to be capable of transforming the measured event times into the microscopic rates of the system including the rates of peptide folding and unfolding. The Gibb's free energy of the folding reaction from these measured rates, 1.7 kJ⋅mol-1 is comparable to that of other helix-forming membrane active peptides measured by isothermal titration calorimetry.
Type Text
Publisher University of Utah
Subject Fluorescence, Microscopy, Raman, Single-molecule, Spectroscopy
Dissertation Institution University of Utah
Dissertation Name Doctor of Philosophy
Language eng
Rights Management Copyright © Grant Aaron Myers 2012
Format Medium application/pdf
Format Extent 10,238,901 bytes
ARK ark:/87278/s6jm2rfc
Setname ir_etd
Date Created 2012-10-11
Date Modified 2016-07-06
ID 195609
Reference URL