Figure 1.

Amide ¹H and ¹⁵N relaxation dispersion profiles have been recorded on a complex comprising the single stranded H2A.Z-H2B protein and the chaperone Chz.core (residues 71–132 of Chz1), prepared as described previously¹⁷. Both protein constituents were U-¹⁵N, ²H labeled. The NMR sample was prepared with [H2A.Z-H2B]_Total≈1 mM, [Chz1]_Total ≈1 mM and 25 mM MES, 0.2 M NaCl, 1 mM EDTA, 10% D₂O, pH = 6.0. Representative ¹⁵N relaxation dispersion profiles from Ala124 of Chz1 (a) and Glu 75 of H2A.Z (b) recorded at static magnetic field strengths of 18.8 T (blue) and 11.7 T (red), 35°C are shown. The vertical lines associated with each measurement (circle) are error estimates; error values of 1% of R₂, 0.3 s⁻¹ or the standard deviation (SD) of duplicate measurements (whichever is the largest) were assigned to the ¹⁵N rates, while errors in ¹HN transverse relaxation rates were based on maximum(3% of R₂, 0.75 s⁻¹, SD of duplicate measurements). The solid lines are fits of a two-site exchange model to the data: |Δϖ|=2.3±0.1 ppm for Ala124(Chz), |Δϖ|=1.8±0.1ppm for Glu75(H2A.Z). Exchange rates and populations are given in the text. (c) Residue specific Δϖ_RMS values calculated as described in the legend to Figure 2. The CHZ motif (residue 95–115) is highlighted with a green bar. (d) Correlation plot of ¹⁵N chemical shift differences for Chz1 measured in a CPMG relaxation dispersion experiment, Δϖ_CPMG,N, versus the difference between the assigned chemical shifts of the bound state of Chz1 and random roil values²⁷, Δϖ_RC,N. Relaxation dispersion profiles were recorded using Varian Inova NMR spectrometers operating at static magnetic field strengths of 11.7 T and 18.8 T (500 and 800 MHz ¹H frequency), respectively. Constant-time²² relaxation compensated²⁰ TROSY-based pulse schemes were used for recording ¹H ³⁴ and ¹⁵N ²¹ relaxation dispersion profiles. A constant-time relaxation delay of 25 ms (40 ms) was used for the proton (nitrogen) dispersion profiles. Effective ¹⁵N transverse relaxation rates (R_2,eff) were measured for 14 different ν_CPMG frequencies between 50 and 1000 Hz, while the proton dispersion profiles were sampled at 15 different ν_CPMG frequencies between 80 and 1840 Hz. Data sets were processed with the NMRPipe program³⁵ and signal intensities quantified using the program FuDA (flemming@pound.med.utoronto.ca; http://pound.med.utoronto.ca/software). Relaxation dispersion profiles, R_2,eff(ν_CPMG), were generated from peak intensities, I(ν_CPMG), in a series of 2D ¹HN-¹⁵N correlation maps measured as a function of CPMG frequency (ν_CPMG=1/2τ, where τ is the time between two successive refocusing pulses). Peak intensities were converted into effective relaxation rates via R_2,eff= ln[I₀/I(ν_CPMG)]/T_relax, where I₀ is the peak intensity in a reference spectrum recorded without the constant-time relaxation delay T_relax. Details of the procedure by which dispersions profiles are fit are provided in the legend to Figure 3.