Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer |
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Affiliation: | 1. Department of Physiology & Biophysics, Stony Brook University, Stony Brook, NY 11794-8661, USA;2. Department of Physics and Astronomy, Clemson University, Clemson, SC 29634-0978, USA;3. National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, CT 06269, USA;4. Department of Molecular and Cell Biology, and Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA |
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Abstract: | Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment. |
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Keywords: | Fluorescent protein FRET Supertertiary structure Discrete molecular dynamics Macromolecular crowding AUC" },{" #name" :" keyword" ," $" :{" id" :" k0035" }," $$" :[{" #name" :" text" ," _" :" analytical ultracentrifugation CHO" },{" #name" :" keyword" ," $" :{" id" :" k0045" }," $$" :[{" #name" :" text" ," _" :" Chinese hamster ovary cells CLY" },{" #name" :" keyword" ," $" :{" id" :" k0055" }," $$" :[{" #name" :" text" ," _" :" CFP-linker-YFP DMD" },{" #name" :" keyword" ," $" :{" id" :" k0065" }," $$" :[{" #name" :" text" ," _" :" discrete molecular dynamics eCFP" },{" #name" :" keyword" ," $" :{" id" :" k0075" }," $$" :[{" #name" :" text" ," _" :" enhanced cyan fluorescent protein eYFP" },{" #name" :" keyword" ," $" :{" id" :" k0085" }," $$" :[{" #name" :" text" ," _" :" enhanced yellow fluorescent protein FRET" },{" #name" :" keyword" ," $" :{" id" :" k0095" }," $$" :[{" #name" :" text" ," _" :" fluorescence resonance energy transfer FP" },{" #name" :" keyword" ," $" :{" id" :" k0105" }," $$" :[{" #name" :" text" ," _" :" fluorescent protein IDR" },{" #name" :" keyword" ," $" :{" id" :" k0115" }," $$" :[{" #name" :" text" ," _" :" intrinsically disordered region PEG" },{" #name" :" keyword" ," $" :{" id" :" k0125" }," $$" :[{" #name" :" text" ," _" :" polyethylene glycol PMT" },{" #name" :" keyword" ," $" :{" id" :" k0135" }," $$" :[{" #name" :" text" ," _" :" plasma membrane targeted RMS" },{" #name" :" keyword" ," $" :{" id" :" k0145" }," $$" :[{" #name" :" text" ," _" :" root mean squared SDS-PAGE" },{" #name" :" keyword" ," $" :{" id" :" k0155" }," $$" :[{" #name" :" text" ," _" :" sodium dodecyl sulfate–polyacrylamide gel electrophoresis SD" },{" #name" :" keyword" ," $" :{" id" :" k01655" }," $$" :[{" #name" :" text" ," _" :" standard deviation SEC" },{" #name" :" keyword" ," $" :{" id" :" k0165" }," $$" :[{" #name" :" text" ," _" :" size exclusion chromatography SAW" },{" #name" :" keyword" ," $" :{" id" :" k01653" }," $$" :[{" #name" :" text" ," _" :" self-avoiding random walk TIRF" },{" #name" :" keyword" ," $" :{" id" :" k0185" }," $$" :[{" #name" :" text" ," _" :" total internal reflection fluorescence illumination |
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