7). This gave Kapo values of 1.3013 M-1 and 2.9012 M-1 for wild-type and V66A/L68V CzrAs, respectively, at 0.23 M NaCl. KZn was measured by direct titration at 0.23 M NaClJ Mol Biol. Author manuscript; available in PMC 2014 April 12.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCampanello et al.Page(see Fig. 1d) and Gc was then calculated from, Gc=-RTln(KZn/Kapo). In order to estimate the associated error on Gc, minimal and maximal Gc values, Gc,min and Gc,min, respectively, were calculated by first determining Kapomin and Kapomax by incorporating the associated errors on the slope and y-intercept obtained from the linear fit of log Kapo versus log [NaCl] for wild-type and V66A/L68V CzrAs (see Supplementary Figure 7). KZnmin and KZnmax were determined by using the standard error of the fit giving Gc,min= -RTln(KZnmax/Kapomin) and Gc,max = -RTln(KZnmin/Kapomax). The standard error reported for Gc, is the average error of the difference of Gcmax minus Gc and Gc minus Gcmin for wild-type and V66A/L68V CzrAs. For all other single mutants, Kapo was calculated as the average value of the extrapolated values for Kapo for wild-type and V66A/ L68V CzrAs at 0.23 M NaCl (8.0 012 M-1). Gc and the associated error was then calculated in the same manner, except Kapomin= Kapo,V66A/L68V and Kapomax=Kapo, WT. NMR spectroscopy NMR spectra were acquired on a Varian VNMRS 600 MHz spectrometer equipped with a cryoprobe in the METACyt Biomolecular NMR Laboratory at Indiana University. NMR samples contained 0.25 mM 15N-labeled H96C CzrA or 0.07 mM 15N-labeled H97MeH CzrA in 10 mM d13-MES, 50 mM NaCl and 2 mM DTT with or without 1.1 monomer mol equiv of Zn(II) added (pH 6.0). 1H5N HSQC or TROSY spectra were acquired at 40 as described previously.35 All spectra were processed and analyzed using NMRPipe and SPARKY with resonance assignments made by inspection. Uniformly 13C, 15N-labeled V66A/L68V CzrA was prepared as previously described for wild-type CzrA, and residuespecific backbone assignments of Zn2 V66A/L68V CzrA were obtained in 10 mM d13-MES, 50 mM NaCl, pH 6.0 using standard triple resonance methods. X-ray crystallography V66A/L68V CzrA was extensively dialyzed into 10 mM Hepes, 50 mM NaCl, pH 7.0. The calculated protein concentration after dialysis was 460 . The protein stock was loaded 1:1 with Zn(II), and crystallized under conditions of 100 mM CHES (pH 9.5), 200 mM NaCl, 10 PEG-8000 (Wizard I, Emerald Biosystems) by hanging drop diffusion at 20 .Sevelamer hydrochloride Diffraction data were collected at -160 on an R-AXIX IV+ detector at Indiana University.Zidebactam All data were processed with HKL2000,68 and phase calculations were performed using the PHENIX AutoMR module.PMID:24516446 69 The Zn(II)-bound wild-type CzrA structure was used as a molecular replacement model,28 and an initial refinement model was produced using the PHENIX AutoBuild module.69 Model building was conducted using Coot70 and subsequent refinement models in PHENIX (see Supplementary Table 3 for structure statistics). Isothermal titration calorimetry ITC experiments were carried out using a MicroCal VP-ITC calorimeter using 1.61 mM Zn(II) as titrant in the syringe and solution conditions of 50 mM Hepes, 3.0 mM NTA (Zn(II)CzrA) or 1.0 mM NTA (Zn(II)CzrA zrO) as a Zn(II) competitor, 0.40 M NaCl, pH 7.0, 25.0 , 300 protein dimer, or 38 complex. A self-complementary 28mer DNA was synthesized (MerMade 4) based on the native czr operator sequence (5’TAACATATGAACATATGTTCATATGTTA) annealed.