Circular Dichroism Spectroscopy
An Applied Photophysics Chirascan Circular Dichroism (CD) spectrophotometer was used to acquire CD spectra of wild-type and mutant forms of PlzA. All measurements were made in a temperature-regulated sample holder at 25°C in 10 mM sodium phosphate buffer, pH 7.0, using a 1-mm pathlength quartz cuvette. Prior to measuring CD spectra, protein samples were diluted to approximately 20-25 µM and their UV-Vis spectra measured using a Beckman DU 800 UV-Vis spectrometer. An extinction coefficient of 14,900 M-1 cm-1 at 280 nm, determined by the method of Pace and coworkers (62) using the Expasy ProteinParam tool (63) was used to evaluate concentration from the UV spectra. Samples were diluted fivefold further to concentrations ranging from 4.2-5.5 µM so that high-quality spectra could be obtained down to 180 nm without saturating the CD detector. Spectra were collected from 260 nm to 180 nm using a 1-nm bandwidth, a 1-nm step size and 5 s sampling time per point. Ellipticity was converted to Molar Residue Ellipticity (MRE) using equation 1, where θ is the ellipticity in degrees, l is the pathlength in cm, c is concentration in mol/L and n is the
\(MRE=\left(\frac{100\times\theta}{l\times c\times n}\right)\)(Eq. 1)
number of residues in the protein (261 for PlzA). The STRIDE webserver (64) was used to evaluate secondary structure in the PDB file for PlzA (7MIE). The DichroWeb server (65) was used to analyze the CD data using a truncated (190-240 nm) version of the SP175 reference set (66) and the CDSTTR method (67). The normalized root mean squared deviation NRMSD between the experimental and the spectra calculated by CDSTTR with the SP175 reference set ranged from 0.019-0.024.