Figure 4
The PIFE ruler in ALEX.
(A) Schematic of a dsDNA template containing a protein binding site of the restriction enzymes BamHI and EcoRV positioned in R1 = 1, 2, 3, 5 and 7 bp distance from donor fluorophore Cy3. The acceptor ATTO647N is positioned at the opposite 5′-end in R2 = 40 bp distance. (B) 2D histograms of 40 bp-dsDNA: free dsDNA (black circle) and dsDNA bound to 500 nM BamHI with PIFE (green, circle) in 1, 2 and 5 bp distance. 2D Gaussian fitting was applied to characterize the observed populations; black/green circles mark the FWHM of each distribution. (C) BamHI-bound 40 bp-dsDNA: one dimensional projections of S(EPR) data and 2D Gaussian fits and their centre positions (free form, black; protein-bound form, green). (D) Absolute changes in S(EPr) as function of R1 between Cy3 and BamHI (left) or EcoRV (right). Note that the control experiment with Cy3B (square) showed only little change of S(EPr) compared to Cy3 (triangle) even at close proximity of Cy3B and protein of 1 bp.
