Origin version 7

Desalting and buffer exchange of purified protein were carried out using Sephadex G-25 column (GE Healthcare) equilibrated with the ITC buffer containing 200 mM Tris-HCl, 200 mM NaCl, and 5% glycerol (pH 8.0). Microcal VP-ITC system (Malvern Instruments) was used to conduct the ITC measurements under aerobic or anaerobic conditions. The anaerobic experiments were carried out inside the COY anaerobic chamber. For αMTrp, a total of 45 injections with a reference power of 15 μcal/s and a stirring speed of 155 rpm were performed after the temperature was equilibrated to 20 °C. ITC measurements for L-Trp binding to TDO were carried out with identical settings except injection volumes and the number of injections. The variant enzyme required higher concentration of L-Trp or αMTrp to reach saturation in the ITC experiment. The ITC data were processed and analyzed using non-linear least square curve fitting with Origin version 7.0 (OriginLab Corp.) software by using one-site or two-site binding models.
Psychological evaluation was done using the Center for Epidemiological Studies in Depression Scale (CES-D) [17 ], Sf-36v2 Health Survey (Optum Inc.), the Pittsburgh Sleep Quality Index [18 (link)], and the Psychiatric Diagnostic Screening Questionnaire [19 (link)]. Informed consent for this investigation was obtained from the proband and her parents.

ITC experiments were carried out as described.^{38 (link)} Briefly, enzyme was dialyzed extensively against 20 mM HEPES, pH 8.0, 300 mM NaCl, 5% glycerol, 0.5 mM tris(2-carboxyethyl)phosphine) supplemented with 500 μM MnCl₂ and 10% (v/v) DMSO, and the dialysate was used to prepare fresh working solutions of various inhibitors. The manganese ion was utilized instead of iron since it would not oxidize during the time frame of these experiments. Titration was carried out at 0.42–1.1 mM compound [I] concentration and enzyme [E] concentration of 25–80 μM. Compounds were injected into protein or buffer (to measure the heat of dilution) in 25 steps of 4 μL volume using a MicroCal Auto-iTC₂₀₀. Binding affinity (K_D), stoichiometry (N), and binding enthalpy (ΔH) were determined by fitting the data using the ITC data analysis module of Origin version 7.0 (OriginLab Corp.).
For each ITC experiment, we performed a test run to figure out the optimal enzyme and inhibitor concentrations to be used and range of titrations. Each curve is fitted for 20 data points. Due to the small differences in the K_D values of very similar chemical compounds, the data presented resulted from experiments done at the same time using the same batch of enzymes, which is essential to avoid the variation from batches of enzyme preparation.

A Microcal VP-ITC system (Malvern Instruments) was used to conduct the ITC measurements as previously described.^{62 (link)} The titration buffer consisted of 100 mM KPi with 50 mM NaCl at pH 8. L-tyrosine or analogs were prepared in titration buffer and injected to the cell containing 2 mL of 100–120 μM purified enzyme. Binding of L-tyrosine and its analogs was assessed in a total volume of 300 µL at the following concentrations: 1.5 mM 1, 10 mM 2 and 20 mM 3. After the temperature was equilibrated to 20 °C, a total of 50 injections were performed with a reference power of 15 μcal/s and a stirring speed of 350 rpm. The ITC data were processed and analyzed using non-linear least squares curve fitting of one-site binding models with Origin version 7.0 (OriginLab Corp.) software.

For FRET assay, CFP-UHRF1-YFP wild-type and mutant proteins were adjusted to 0.2 μM, titrated with increasing amount of a hemimethylated DNA duplex (upper strand: 5′-GGCCCXGCAGGCCTG-3′; lower strand: 5′-CAGGCCTGCGGGGCC-3′; X = 5-methylcytosine), USP7 UBL domains or histone peptides. Assays were performed in Take3-plate (#259913 BioTek Instruments) in a buffer containing 25 mM Tris (pH 7.5), 100 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine (TCEP). Emission intensities were scanned using a SynergyMX microplate reader (#255439 BioTek Instruments) from 470 to 600 nm, with 450 nm as the excitation wavelength. The FRET efficiency ratio was calculated as fluorescence intensity at 525 nm divided by the sum of fluorescence intensities at 480 nm and 525 nm. Emission spectra were normalized to the sum of the fluorescence peak intensities at 480 nm and 525 nm. All assays were performed in duplicate, and the data were processed and normalized using Origin version 7.0 (OriginLab).