Browsing by Subject "molecular brightness"

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    Single-molecule studies of novel, genetically encoded, hetero-FRET sensors to environmental ionic-strength using different modalities of fluorescence correlation spectroscopy (FCS)
    (2020-08) Kay, Taryn
    A living cell is a complex environment with heterogeneous and dynamic distributions of ionic strength and macromolecular crowding. Ionic strength influences many aspects of the biology of living cells such as catalytic activities of enzymes, cell volume, osmosis, and protein functions. The challenge, however, is that the ionic strength varies, both spatially and temporarily, throughout the milieu of living cells. As a result, there is a need for ionic-strength sensors that can be genetically encoded in different compartments in living cells, while being amenable to quantitative and noninvasive analytical methods. Importantly, low-level expression of those potential ionic-strength sensors is desirable such that they will not interfere with the function and biological activities of the native protein. In this project, we investigate a family of genetically encoded ionic-strength sensors (mCerulean3-linker-mCitrine) that consist of a donor (mCerulean3), an acceptor (mCitrine), and a linker region made of two oppositely charged α-helices at the single-molecule level. We hypothesize that as ionic strength in the environment increases, the electrostatic attraction between the charged helices will decrease, pulling the donor and acceptor apart, and therefore decreasing energy transfer efficiency at the single-molecule level. To test this hypothesis, we have developed a new approach based on the molecular brightness of the cleaved and intact sensors (RD and KE) for FRET analysis using fluorescence correlation spectroscopy (FCS) in 10 mM sodium phosphate buffer as a function of the environmental ionic strength using different salts. Towards these goals, we rebuilt, calibrated, and optimized a home-built FCS setup, which was used to laser wavelength dependent studies of the fluorescence fluctuation autocorrelation analysis of these sensors. In addition, we characterized the translational diffusion coefficient and hydrodynamic radius of these sensors under different laser wavelengths and compared our results using theoretical model that relate the hydrodynamic volume with the molecular weight of proteins. Our single-molecule approach for FRET analysis of genetically encoded donor-acceptor pairs are particularly amenable to live cell studies with the added advantage of requiring very low expression levels of the sensor as compared with conventional, ensemble-based methods.