Protein expression and purification
Expression and purification protocols for [U-13C,15N] labeled HdeA have been detailed in a previous publication. In order to eliminate chemical shift variation due to changes in buffer type, all samples for the pH titration experiments utilized 50 mM citrate buffer, 10% 2H2O and 0.4 mM DSS (2,2-dimethyl-2-silapentane-5-sulfonate, for chemical shift referencing), unless otherwise stated. For each pH in the titration the protein sample was exchanged into the new buffer using a spin column (2 mL Zeba spin column, 7 kDa MWCO, Thermo Scientific).
All experiments were recorded at 25°C on an Agilent DD2 600 MHz NMR spectrometer with a room-temperature probe. Sample concentrations ranged from ∼1.0 to 1.7 mM. Data were processed using NMRPipe/NMRDraw v.7.2[17, 18] and spectra were analyzed with NMRViewJ v.9.0.[19, 20] Curve fitting was performed using NMRViewJ and QtiPlot.
1H-15N HSQC spectra were recorded on HdeA at pH 3.0, 3.4, 3.7, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.5, and 6.0 to monitor backbone amide chemical shift change. HNCaCb and CbCa(CO)NH chemical shift assignment experiments were also recorded at pH 3.0, 4.0, 5.0, and 6.0 in order to ensure proper tracking of the movement of each assigned peak. Weighted averages of the overall backbone amide (1HN and 15N) chemical shift changes between pH 6.0 and 3.0 were calculated using Eq. (1):
3D TOCSY (C(CO)NH) spectra were recorded at pH 2.6 and 2.8 in addition to the above listed pH values to measure Asp and Glu pKas. Titration curves (from plots of chemical shift as a function of pH for each residue) were fit to a modified Henderson-Hasselbalch equation:
where a and b are the minimum and maximum chemical shift values for the curve, respectively, and n is the Hill coefficient. When fitting data to obtain pKas, the pH values were corrected by 0.04 units to account for the 10% 2H2O in solution.
Hydrogen exchange experiments were performed by rapid exchange of an HdeA sample from citrate buffer in 1H2O into 100% 2H2O. At each pH, 72 x 1H-15N SOFAST HSQC spectra were recorded sequentially, with each spectrum running for 9 min 37 s. Decay curves were fit with a standard exponential decay equation to obtain the observed rate of exchange for each amide group (kobserved), and protection factors were obtained using the equation PF = (kintrinsic/kobserved), where kintrinsic values were obtained from estimations calculated by the online program SPHERE.[24, 25]
Backbone 15N longitudinal (R1) and transverse (R2) relaxation rate constants were recorded on HdeA at pH 3.0, 4.0, 5.0, and 6.0 using standard 1H-15N correlation experiments. R1 values were determined from 14 spectra with eleven delays of 0.01 (x3), 0.05, 0.10, 0.15, 0.20 (x2), 0.25, 0.30, 0.40, 0.50, 0.60, and 0.80 s. R2 values were obtained from twelve spectra with nine delays of 0.01 (x2), 0.03 (x2), 0.05, 0.07, 0.09 (x2), 0.11, 0.13, 0.15, and 0.17 s. The correlation time (τc, in s) at each pH was estimated from 10% trimmed, averaged R2/R1 ratios using Eq. (3):
where νN is the 15N resonance frequency (60 786 494 Hz, in our case). The equation was derived from Kay et al. and the NESG Wiki online. HydroPro was used to calculate the expected correlation time, based on a “rebuilt” crystal structure: since the reported crystal structure (1DJ8) was missing 8 amino acids from the N-terminus and 2 from the C-terminus, we used Swiss-Pdb Viewer to build the missing residues onto the structure and then perform a basic minimization in order to provide a rough structure with a size more comparable to the full-length dimer that could be used for modeling.