DNA and nucleosomes direct distinct folding of a linker histone H1 C-terminal domain.
We previously documented condensation of the H1 CTD consistent with adoption of a defined structure upon nucleosome binding using a bulk FRET assay, supporting proposals that the CTD behaves as an intrinsically disordered domain. In the present study, by determining the distances between two different pairs of sites in the C-terminal domain of full length H1 by FRET, we confirm that nucleosome binding directs folding of the disordered H1 C-terminal domain and provide additional distance constraints for the condensed state. In contrast to nucleosomes, FRET observed upon H1 binding to naked DNA fragments includes both intra- and inter-molecular resonance energy transfer. By eliminating inter-molecular transfer, we find that CTD condensation induced upon H1-binding naked DNA is distinct from that induced by nucleosomes. Moreover, analysis of fluorescence quenching indicates that H1 residues at either end of the CTD experience distinct environments when bound to nucleosomes, and suggest that the penultimate residue in the CTD (K195) is juxtaposed between the two linker DNA helices, proposed to form a stem structure in the H1-bound nucleosome.
PubMed ID: 22021384
PMC ID: PMC3287190
Article link: Nucleic Acids Res.
Grant support: GM52426 NIGMS NIH HHS , Intramural NIH HHS
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|Figure 1. The H1 CTD is intrinsically disordered and condenses upon binding to nucleosomes. (A) Schematics of H1 G0101C/K195C (left) and H1 G101C/T173C (right) modified with Cy3 and Cy5 (red and blue). N, G and C denote the N-terminal, globular and C-terminal domains, respectively. (B) Binding of Cy3/Cy5-labeled H1 G101C/K195C to nucleosomes results in significant FRET. Emission spectra of 5 nM free H1 (black line) and H1 in the presence of increasing amounts of 207 N nucleosomes, as indicated. Numbers indicate molar ratio of nucleosome:H1. Excitation was at 515 nm. (C) As in (B) except protein was Cy3/Cy5-labeled H1 G101C/T173C. (D) Plot of FRET efficiency as function of nucleosome:H1 ratio for Cy3/Cy5-labeled H1 G101C/K195C and H1 G101C/T173C (filled black triangles and blue circles, respectively, Cy3/Cy5). Also shown are efficiencies for 1:1 mixtures of Cy3- and Cy5-only labeled G101C/K195C and G101C/T173C (triangles and circles, respectively, Cy3+Cy5).|
|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.|
|Figure 3. Intermolecular FRET between combinations of labeled sites in H1 upon binding to naked DNA. Pairs of the single substitution mutants H1 G101C and H1 K195C labeled with either Cy3 or Cy5 were mixed in a 1:1 ratio and emission spectra recorded before (black trace) or after (red trace) addition of 207 bp naked DNA fragments. Emission was at 515 nM. (A) H1 G101C-Cy3 + H1 G101C-Cy5. (B) H1 G101C-Cy3 + H1 K195C-Cy5. (C) H1 K195C-Cy3 + H1 K195C-Cy5. The red arrow indicates the Cy5 emission peak. Numbers above the arrow indicate relative FRET efficiency in each experiment.|
|Figure 4. Nucleosome-induced fluorophore quenching is dependent on attachment site. (A) Emission spectra of Cy5-modified H1 G101C were recorded in the absence and presence of increasing concentrations of nucleosomes, as indicated. (B) As in A except Cy5-H1 K195C was used. (C) Plots of the extent of quenching as a function of the nucleosome:H1 ratio. Quenching was determined by the peak height compared to that in the absence of nucleosomes (see ‘Materials and Methods’ section).|
|Figure 5. Quenching due to H1 binding naked DNA. (A) Quenching of Cy5-H1 G101C fluorescence depends on binding mode. The protein was incubated with increasing amounts of 207 bp naked DNA fragment and the fraction of Cy5 fluorescence quenched determined as compared to fluorescence in the absence of DNA and plotted. Quenching in buffers containing 50 or 20 mM NaCl (cooperative and non-cooperative conditions, respectively) is shown. (B) As in A except Cy5-H1 K195C was used.|