Research: Atomic Force Microscopy, Past Studies, Single Molecule FRET
Topic: DNA Mismatch Repair
AFM is a powerful technique for studying protein-protein and protein-DNA interactions. However, a significant limitation to AFM and other high-resolution microscopy is the difficulty of identifying different proteins in multi-protein complexes. To resolve this, we are interested in combining the power of AFM and fluorescence microscopy techniques.
High accuracy FIONA–AFM hybrid imaging
We developed FIONA-AFM, which combines FIONA (fluorescence intensity with one nanometer accuracy) and AFM to achieve both high resolution and non-ambiguous resolving capability among different molecules.
Combining AFM with FRET
We are developing a combination of fluorescence resonance energy transfer (FRET) and atomic force microscopy (AFM) to enable identification of specific proteins within the complexes. The protein of interest is fluorescently labeled with the acceptor dye of a FRET pair, and the corresponding FRET donor is immobilized on the AFM tip.
FRET occurs when acceptor and donor come close together. The strong distance dependency of FRET can be exploited to preserve high spatial resolution. The FRET signal is measured at each sample position scanned by the tip using an avalanche photodiode (APD) and then superposed with the AFM topography image. The identification of the individual protein components will allow us to determine the relative positions and stoichiometries of the proteins, and to visualize small protein molecules next to large proteins that would otherwise be invisible to the AFM.
The technique will be applied to the study of biologically essential DNA repair complexes, consisting of several different proteins.
Figure. Schematic diagram of FRET-AFM
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Image credit: Ingrid
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