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.