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Fig. 2.
Emission scan of the donor and the acceptor in conformationally changed MBP in response to maltose. (A) Maltose-induced conformational changes in MBP were evaluated via intramolecular FRET analysis with an LS55 Luminescence Spectrometer (Perkin–Elmer Instruments) from ECFP:MBP:EYFP samples at varying incubation times of maltose (0, 5, 10, 20, and 30 min). The excitation wavelength was set to 433 nm, and the emission was subsequently measured in Hesperadin range of 460–570 nm. All reactions were conducted at 25 °C with 10 mM maltose. (B) Glucose was utilized as a negative control ligand under the same experimental conditions.
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FRET acceptor photobleaching for measuring maltose-induced conformational change in MBP
Fig. 3.
FRET acceptor photobleaching. The EYFP acceptor was bleached using a confocal laser-scanning microscope at 514 nm. The fluorescence emissions of the donor and the acceptor were analyzed from E. coli cells expressing the ECFP:MBP:EYFP protein in the presence and absence of maltose using the ‘multitrack’ function of the Zeiss LSM 510 software.