Browsing by Subject "z-scan"

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    Brightness analysis in finite geometries: probing protein interactions in cellular, cell-free and aqueous environments
    (2012-12) Macdonald, Patrick
    Fluorescence fluctuation spectroscopy (FFS) is a powerful technique for quantitatively analyzing protein interactions. Using brightness analysis methods, we are uniquely able to measure the stoichiometry of protein complexes. FFS is particularly valuable because it allows measurements within living cells. This thesis demonstrates that measuring in very small volumes, such as E. coli cells, introduces a bias into the measured brightness. We show that this bias is a result of accumulative sample loss, or photodepletion, and that we can account for this effect and recover correct brightness values. Similarly, very thin samples, such as cell cytoplasm, introduce a bias due to the sample being shorter along the vertical axis than the volume of the excitation light. We introduce z-scan FFS and theory to identify and model thin samples and to recover unbiased data. Although measuring in cells is a primary strength of the FFS technique, some studies require the greater degree of experimental control afforded by solution measurements. Thus, we characterize cell-free expression solution for FFS measurements, an environment that offers increased control but permits genetic fluorescent labeling. We take advantage of this system to perform chromophore maturation experiments as a function of temperature on three common fluorescent proteins: EGFP, EYFP and mCherry. Our results prove that EGFP has fast maturation and is a good reporter for fluorescence experiments. Finally, we apply FFS and brightness analysis to the enzyme, APOBEC3G. We reveal that APOBEC3G interactions with RNA and single-stranded DNA are sequence dependent, which has important implications for the mechanism by which APOBEC3G packages itself into HIV-1 viral particles and restricts the virus to prevent infection.
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    Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins
    (2015-05) Smith, Elizabeth
    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.