Figure 2. Spin-labeled ATP analogues reveal conformational changes in the ATP-binding pocket of SNF2h.

a. 2′3′SLATP supports SNF2h nucleosome remodeling. Remodeling kinetics of N+80 nucleosomes (5 nM) with 150 nM of SNF2h, 1mM MgCl₂ and 0.5mM of ATP or 2′3′SLATP (k_obs for n=3 is 2.0±0.06 min⁻¹ and 1.2±0.22 min⁻¹, respectively for ATP and 2′3′SLATP). Representative remodeling traces are shown. Inset: 2′3′SLATP analog (nitroxyl-radical spin probe shown in red). b. Representative EPR spectra of 2′3′SLADP and 2′3′SLADP-BeF_x with SNF2h and N+60 nucleosomes. The spectrum of 2′3′SLADP alone (without SNF2h and nucleosomes) is shown in black. The presence of nucleosomes and SNF2h (pink spectrum) produces a bound peak (P1) in the 2′3′SLADP spectrum with a high-field to low-field (P1–P5) splitting of 4.8mT (118.8° cone angle). When the analog 2′3′SLADP•BeF_x is used with nucleosomes and SNF2h (blue spectrum) this produces a second, more immobilized peak (P1, blue) with a P1–P5 splitting of 6.2mT (68° cone angle). Spectral peaks are truncated to enhance resolution of bound probe. c. Representative spectra of 2′3′SLADP (pink) and 2′3′SLADP•BeF_x (blue) bound to SNF2h in the presence saturating concentrations of a 60 base pair DNA fragment. The black spectrum (2′3′SLADP alone) is from panel b for comparison. d. Comparison of the ATPase rates of wildtype enzyme (black) and the E309A mutant (blue) in the presence of N+60 nucleosomes. The curves level off due to depletion of MESG, the Enzchek assay detection reagent (See Methods). e. Representative spectra of 2′3′SLADP (pink) and 2′3′SLADP•BeF_x (blue) bound to the E309A SNF2h mutant in the presence of N+60 nucleosomes.