Encapsulation of fluorescent proteins in folded-sheet mesoporous materials: Effect of pore size on energy-transfer efficiency

The assembly and relative orientation of two fluorescent proteins (sGFP and DsRed) encapsulated in four kinds of mesoporous silica – namely, ethanol-treated folded-sheet mesoporous materials (FSMs) with different pore sizes (i.e. pore diameters of 2.6, 3.7, 5.4, and 7.1 nm) – were confirmed directly using a fluorescence resonance energy-transfer (FRET) technique. The physicochemical properties of the ethanol-treated FSMs were analyzed finely by powder X-ray diffraction, and nitrogen adsorption/desorption measurements. When sGFP and DsRed were encapsulated in the FSM mesopores, the sGFP–DsRed–FSMs composites showed a notable FRET signal, indicating that the composites function as an energy-transfer system through the combination of the two proteins, due to the successful encapsulation of the sGFP–DsRed pairs in the pores. The FRET signal indicates the possibility of a high level of accessibility between sGFP and DsRed, as encapsulated in the same FSM channels. Interestingly, the FRET efficiency depended highly on the FSM pore size. In the case of the protein concentration of 1 and 2 mg/mL, FRET efficiency reached to a maximum value at a pore diameter of 3.7 nm (FSM3.7). In contrast, the proteins encapsulated in FSM2.6 (i.e. pore diameter of 2.6 nm), which would arrange both sGFP and DsRed tandemly along the one-direction pores, indicated a higher FRET efficiency at 4 mg/mL. These results strongly demonstrated that FRET efficiency depends on not only the distance between the proteins, but also the relative orientation of the proteins encapsulated in the pores.