All NMR spectroscopy experiments were recorded at 298 K on a 14.1-T Varian INOVA spectrometer equipped with a 5-mm z-axis gradient triple resonance room temperature probe. Spectra were processed with NMRPipe and analyzed with NMRFAM-Sparky. Two-dimensional sensitivity-enhanced 1H-15N heteronuclear single-quantum coherence (HSQC) spectra were acquired with 1H (15N) 512 (64) complex points, spectral widths of 8012 Hz (1800 Hz), maximum acquisition times of 64 ms (35.6 ms), an interscan delay of 1 s, and four scans per free induction decay for a total acquisition time of 19 min. 13C,15N-2PHspB177–171 was prepared at a final concentration of 0.9 mM in 30 mM sodium phosphate, 2 mM EDTA (pH 7.0) for resonance assignments, wherein HNCA, HNCO, and HN(CA)CO spectra were recorded. The assigned 1HN, 15N, 13CO, and 13Cα chemical shifts were analyzed with TALOS-N and RCI to respectively estimate the secondary structure and N-H order parameters.

Protonated, 15N-labeled proteins were prepared at 0.9 mM (2PHspB177–171) or 1 mM (HspB184–171) for 15N relaxation studies. Standard pulse sequences to measure transverse relaxation times (T2), 15N heteronuclear nuclear Overhauser enhancements (hetNOE), and 15N CPMG (Carr-Purcell-Meiboom-Gill sequence) relaxation dispersion were used. The T2 experiments contained eight delay times up to 154 ms and an interscan delay of 2 s. The intensity changes over time (I/I0) were fit to an exponential decay, and values are reported as rates (i.e., 1/T2) for convenience. hetNOE experiments contained an interscan delay of 8 s, with values representing the intensity upon amide proton saturation divided by the intensity in its absence. CPMG relaxation dispersion experiments were recorded with variable delays between 180° pulses in the CPMG pulse train (τCPMG = 4νCPMG−1). A constant relaxation delay of 39 ms for the CPMG period and 20 νCPMG values ranging from 54 to 950 Hz were used. Peak shapes were fit with FuDA to extract peak intensities, which were then converted into R2,eff values using the following relation: R2,effCPMG) = −1/Trelax ln[ICPMG)/I(0)], where ICPMG) is the intensity of a peak at νCPMG, Trelax is the constant relaxation delay of 39 ms that was absent in the reference spectrum, and I(0) is the intensity of a peak in the reference spectrum. Two duplicate νCPMG points were recorded in each dispersion dataset for error analysis, and uncertainties in R2,eff were calculated using the SD of peak intensities from such duplicate measurements. From plots of R2,eff as a function of νCPMG, Rex was determined by taking the difference of R2,eff (54 Hz) and R2,eff (950 Hz) with error bars representing the propagated errors in R2,eff.

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