A Fluorescence Resonance Energy Transfer Sensor Based on Maltose Binding Protein Xianhui Li 04-13-2004

Outline Background What is a FRET? Incorporating FRET into a MBP-based sensor Application of the sensor in measuring maltose concentration conclusion

Background FRET has been widely used to study many protein-protein, protein-DNA, molecular structure, and other interactions. The use of FRET in carbohydrate analysis has not been fully exploited. Incorporating FRET reporter into a biosensor based on maltose binding protein can potentially achieve high specificity and sensitivity. Measuring carbohydrate levels has many potential uses, such as in bread and beer production, and glucose monitoring for diabetics.

What is a FRET? • Donor and acceptor molecules must be in close proximity (typically 10–100 Å). • The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor Emission Emission

Schematic Representation of the FRET-based Sensing Scheme Donor: a mutant Maltose Binding Protein (MBPD95C) was labeled with Cy3.5. Acceptor: β-cyclodextrin-Cy5 (β-CD-Cy5) adduct

Absorption and emission spectra of MBP-Cy3.5 and β-CD-Cy5 Emission spectrum of MBP-Cy3.5 overlaps absorption spectrum of β-CD-Cy5

FRET process takes place between donor and acceptor components Titration of 0.1uM MBP-Cy3.5 (1dye/protein ratio) with increasing amounts of β-CD-Cy5. Samples were excited at 520nm

Further verify occurrence of FRET between donor and acceptor Fluorescence emission spectra of 0.1uM MBP-Cy3.5(1.7 dye/protein ratio) titrate against increasing amounts of β-CD -Cy5(A) or unlabeled β-CD(C) (1 D/P ratio). 0.5uM β-CD-Cy5 titrated with increasing amount of unlabeled MBP β

Maltose Analysis Increasing the maltose concentration causes a systematic increase in MBP-Cy3.5 donor fluorescence intensity

Conclusion The FRET sensor allows measurement of maltose concentrations through the gain in donor fluorescence intensity. The FRET sensor is very sensitive, with lower detection limits of 50-100nM maltose and upper limits approaching 50uM. (This is at least 20 times more sensitive than the best current method)