4/4_RecombinantDNATechnology_Hoffman 4/4/2017 3:00:00 PM Fluorescent Molecules (GFP) • Will lose their fluorescence if a light is shined on them too much • Also takes a moment for you to be able to see the difference—for the fluorescence to show Where in a cell is “my” protein • Fusion to GFP (Verify if protein is still functional) • Live cell microscopy Do two proteins co-localize? • Are they in the same location in the cell • GFP to one protein • YFP to the second protein • Filters to see the two different emission wavelengths • Seeing them in the same location doesn’t really mean that much though, what we really want to know is if they work together. • … Q: Do my proteins interact or when do they interact? A: FRET • We can add compounds/ molecules/ etc. to interact… see if it changes the function • FRET= fluorescent resonance energy transfer • Can detect when CFP and YFP are within 10 nm of each other o 10 nm= diameter of a typical protein o this gets us down to the level of protein-protein interactions • Protein X- CFP and Protein Y- YFP Excitation Emission GFP 488 507 CFP 434 476 YFP 514 527 • All very different wavelength • For CFP- we give it photons coming in at 434 and we should read emission being emitted at 476 • YFP emission at 527 o The two emissions are too far from each other now in distance • But maybe they interact with each other in different conditions o There is some kind of energy transfer from one protein to the other • If CFP is excited at 434 and YFP is receiving energy, then CFP is losing energy we would see a reduction in the CFP (476) Emission and an increase in the YFP (527) emission o This would show that these two proteins are within the typical distance of a protein-protein interaction o **must excite from the lower wavelength to the higher FRET!: is a mechanism describing energy transfer between two lightsensitive molecules (chromophores). [1]A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. [2]The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in [3] distance. • Measurements of FRET efficiency can be used to determine if [4] two fluorophores are within a certain distance of each other. Another use of FRET- as a sensor = FRET Sensors • Detects changes in protein-protein interaction or a change in the conformation of a protein • FRET based proteins that are known to interact or lose an interaction based on something else going on in the cell (gives a measurement of what is disturbing the interaction in the cell) • Example: Cyclic AMP detection (can be used for calcium detection, any of a number of small molecules that would change a protein- protein interaction or the conformation of a protein) o PKA forms when there are low cAMP levels o When there are high cAMP levels- the regulatory units binds cAMP and changes its shape o This protein-protein interaction is controlled by cAMP levels o One way of detecting cAMP levels is by putting CFP on the regulatory unit and YFP on the catalytic unit o Under low cAMP levels you would see a FRET signal; under high cAMP levels you would see a loss of FRET signal 3 more uses of fluorescence in molecular cell biology FRAP- fluorescence recovers alto photo-bleaching • Continual UV exposure bleaches a FP no longer fluorescent (will not recover fluorescence) • Take a