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Figure 2. Binding of Cy3/Cy5-labeled H1 G101C/K195C to naked DNA results in both intra- and inter-molecular FRET. (A) Binding of Cy3/Cy5 labeled H1 G101C/K195C to naked DNA induces FRET. The protein was incubated alone (black trace) or with increasing amounts of naked 207-bp DNA fragments, as indicated. The molar ratio of DNA:H1 is indicated as is the concentration of DNA in microgram and microliter (in parenthesis). (B) Intermolecular FRET upon H1 binding to naked DNA. A 1:1 mixture of Cy3-only and Cy5-only labeled H1 G101C/K195C was incubated alone or in the presence of increasing amounts of 207-bp DNA fragment as in A. (C) Schematic of H1–DNA ‘tramtrack’ structure (24). H1 is indicated by the red ovals; DNA by the lines. (D) Model for dilution of inter-molecular FRET with unlabeled H1 (open ovals). (E) Elimination of inter-molecular FRET to reveal intra-molecular FRET. Efficiencies for samples prepared as in A (H1-Cy3/Cy5) or B (H1-Cy3 + H1-Cy5) and were determined and plotted (0 point) along with efficiencies for samples in which increasing fractions of the H1 was not labeled with fluorophores. Image published in: Fang H et al. (2012) © The Author(s) 2011. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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