Manipulating protein conformations by single-molecule AFM-FRET nanoscopy

Abstract

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.

Figure 1

(A) Crystal Structure of HPPK. The green spirals represent α helices and the blue arrows represent β strands. The loops are shown by the red pipes. Amino acid residue 88 and 142 has been labeled with FRET dye pair Cy3 and Cy5, respectively. (B) HPPK catalyzed pyrophosphorylation transfer two phosphor groups from ATP to HP.

Figure 2

(A) Single-molecule AFM-FRET ultra nanoscopy, the zoomed panel in the left presents schematic diagram of one FRET dye-pair (donor-acceptor: Cy3–Cy5) labeled HPPK molecule tethered between a glass cover-slip surface and a handle (biotin group plus streptavidin), and another biotin group is modified on AFM tip. (B) Single-molecule fluorescence photon counting images of the donor (Cy3, left) and accepter (Cy5, right). Each feature is from a single HPPK enzyme labeled with Cy3–Cy5 FRET dyes.

Figure 3

(A) A typical FRET time trajectory of donor (green) and acceptor (red) associated with one single-molecule AFM-FRET force pulling event. (B) Zoom-in intensity trajectory of donor and acceptor from (A), the highlighted intensity change is correlated to one pulling event occurred in 0.04s. (C) FRET efficiency time trajectory of one single-molecule AFM-FRET pulling event, in the whole process of AFM tip travelling route from approaching the protein from far away to moving away out of the micro-mirror effect distance range, three efficiency levels are recorded and identified. The error bar shows the ±2SD (standard deviation) indicating ≥ 95% precision of identification of the data points within the range. (D) The correlated force curve, the curve shows the extension length of 24 nm within a period of 0.04 s.

Figure 4

(A) Histogram of extension length distribution of AFM-FRET force unfolding single-molecule proteins. The primary extension length within a range of 20–40 nm, and the mean extension length is about 24–28 nm. (B) Histogram of protein ruptures force distribution. The distribution shows two peaks, and the most probable rupture forces are 16–18 pN and 50–52 pN.

Figure 5

(A–C) Three types of single-molecule force pulling curves of HPPK, as HPPK was chemically linked to a glass coverslip at residue 142, and AFM tip pulling occurs at the possible lysine residue site 119, 85, and 23. In the insets above the force curves, three proposed domains are colored (green for DomA, purple for DomB and red for DomC) and depicted. (A) The unfolding force curve of DomC (red), which corresponds to the rupture distance 9 nm. (B) The unfolding force curves of DomB (purple) and DomC (red), corresponding to the rupture distance 22 nm. (C) The unfolding force curve of DoA, DoB and DoC, and the rupture distance is 45 nm. (D) Histogram of protein rupture distance distribution. The distribution of the rupture distances shows three peaks, at about 9 nm (DomC), 22 nm (DomB and DomC), and 45 nm (DomA, DomB and Dom C). (E) The structure of the HPPK mutant (the site of lysines and cysteine are illustrated). Amino acid residue 142 was mutated to cysteine for specific site tethering of HPPK on the glass cover-slip.