Manipulating Protein Conformations by Single-Molecule AFM-FRET Nanoscopy

Yufan He, Maolin Lu, Jin Cao, and H. Peter Lu*

Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, United States

ACS Nano, Article ASAP

DOI: 10.1021/nn2038669

Publication Date (Web): January 25, 2012

Copyright © 2012 American Chemical Society

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.