Protein Engineering Protocols - Mycobacteriology research center

Design of Zinc Finger Proteins 873.1.4. CD MeasurementThe CD spectra of the zinc finger proteins are recorded on a Jasco J-720 spectropolarimeterin Tris-HCl buffer, pH 8.0, containing 50 mM NaCl in a capped 1-mm path-length cell at 20°C, under nitrogen. All spectra represent the average of 8to 16 scans. Spectra are baseline corrected and noise reduced using Jasco software.3.1.5. UV-VIS Absorption SpectroscopyUV-VIS absorption spectra are recorded on a Beckman Coulter DU7400 diodearray spectrophotometer at 20°C in 10 mM Tris-HCl buffer, pH 7.5, containing 50mM NaCl in a capped 1-cm path-length cell. Co(II)-substituted zinc finger complexesare obtained by titration with CoCl 2. The peptides are saturated with Co(II) inarbitrary conditions. All spectra are normalized by ε = A/(l •c), where ε is the extinctioncoefficient (per molar concentration per centimeter), l is the path-length of thecell (in centimeters), and c is the peptide concentration (in molar concentration).3.1.6. NMR ExperimentsIn the presence of 1.5 Eq of Zn(II) ion, the complex of single finger domainand Zn(II) is prepared at a concentration of 5 mM in 90% H 2O/10% D 2O andD 2O (25 mM Tris–d 11, pH 5.7). All of the NMR spectra are recorded on a JOELLambda-600 spectrometer.1. The nuclear Overhauser enhancement spectroscopy (NOESY) spectra areacquired with selective water presaturation (delays alternating with mutations fortailored excitation pulse) followed by the standard NOESY pulse train at 303 K,with mixing times of 100, 200, and 300 ms.2. The total correlation spectroscopy spectra are obtained with an 80-ms MLEV-17 spinlockduration, using water suppression by a gradient tailored excitation pulse at 303 K.Spectra are typically acquired with 24 scans per t1 valve, for 1024 t1 valves,and 2048 complex points are collected in the direct dimension. The free-inductiondecay in both dimensions is multiplied by the phase-shifted sine bell apodizationfunction, zero-filled, and Fourier transformed to yield 2048 by 2048 matrices.Sequential resonance assignments are determined by standard total correlationspectroscopy and NOESY procedures (8).3.2. Six- and Nine-Zinc Finger ProteinsThis section describes the design strategy and construction of multi-zinc fingersfor the recognition of long DNA sequences.3.2.1. Strategy of Protein DesignNovel six- and nine-zinc finger proteins, Sp1ZF6 and Sp1ZF9, were createdfrom the three-zinc finger motifs of the transcription factor, Sp1 (Fig. 2). These