Based on the theoretical principles of FRET, the objective of this study was to assess the characteristic hinge-twist motion of MBP in response to maltose treatment using intermolecular FRET technology. Upon the addition of maltose, the two lobes of MBP move in close proximity to each other, thereby resulting in a concomitant
GDC-0199 transfer from the donor to the acceptor fluorophores. Accordingly, a downshift in the donor (ECFP) emission intensity is anticipated, followed by a subsequent increase in the acceptor (EYFP) emission (Fig. 1A). In service of this objective, the pECFP:MBP:EYFP plasmid used to generate the chimeric ECFP:MBP:EYFP recombinant protein was genetically constructed (Fig. 1B), as described in the Materials and Methods section. The chimeric ECFP:MBP:EYFP protein was then expressed in E. coli as a recombinant protein extended by a hexahistidine (6 × His), which is
anticodon the most extensively used affinity tag. After IPTG induction, a clear additional band equal to the molecular weight of 94 kDa ( Fig. 1C) was detected, a finding which was consistent with the expected molecular weight of ECFP:MBP:EYFP.
