Manipulating protein conformations by single-molecule AFM-FRET nanoscopy.

Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3-Cy5-labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling.

Citation

Yufan He, Maolin Lu, Jin Cao, H Peter Lu. Manipulating protein conformations by single-molecule AFM-FRET nanoscopy. ACS nano. 2012 Feb 28;6(2):1221-9

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PMID: 22276737

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