Hcn 5 mm triple resonance z axis pfg room temperature probe

The solution NMR samples prepared for pH titrations were made in a volume of 375 µL to account for negotiable sample loss during adjustment of the pH. The pH was monitored before and after the experiment and found to fluctuate by ±0.01. Solution NMR experiments were performed on an Agilent DD2 600 MHz spectrometer with an HCN 5 mm triple resonance z-axis PFG room temperature probe. For the pH titrations, ¹H/¹³C SOFAST-HMQC experiments^{12 (link)} were acquired at 15 °C and 25 °C using a spectral width for ¹H and ¹³C was 12019.2 Hz (83.2 msec acquisition time) and 4000 Hz (27.5 msec, evolution time), respectively and a recycle delay of 1 sec. The complete pH titration curve was collected over ~1–2 days and residues showing significant chemical shift perturbations (¹H > 0.03 ppm and ¹³C > 0.1 ppm) were globally fit using Eqn 1, where δ_HA and δ_A− are the chemical shifts for the protonated and deprotonated transporter, respectively, while δ is the observed chemical shift at the different pH values.
$$\delta =\frac{{\delta }_{HA}+{\delta }_{A-}{10}^{pH-pKa}}{1+{10}^{pH-pKa}}$$
The chemical shift perturbation between protonated and deprotonated EmrE at Glu-14 (Δδ) in the ¹³C (Δδ_C) and ¹H (Δδ_H) dimensions of the methyl spectra were calculated using a ¹³C scaling factor of 0.184 with the equation below.
$$\mathrm{\Delta }\delta =\sqrt{{(0.184\mathrm{\Delta }{\delta }_C)}^2+\mathrm{\Delta }{\delta }_H^2}$$
Methyl T_1zz exchange spectra were acquired with a mixing time 300 ms and a recycle delay of 1 sec.