Biology Reference
In-Depth Information
11.2.1.7.2
Circular dichroism spectroscopy
The transmembrane proteins that we have purified have all had their topologies
determined empirically using glycosylation site mapping, limited trypsin digestion,
and indirect immunofluorescence microscopy; therefore, circular dichroism was
used to determine the predominant secondary structure of the purified recombinant
protein. In order to do this, spectra of purified proteins at a concentration of 3
m
Min
Buffer C were collected on a Jasco J-815 CD spectrophotometer using a 2 mm path
length cuvette at room temperature. Spectra demonstrated that FIT1 and FIT2 are
a
-helical in nature due to the characteristic spectral minima at 206 and 222 nm
(
Fig. 11.3
). Proteins that contain predominantly
b
-sheet character have a single
minimum at 213-215 nm and proteins predominantly consisting of random coils
have a distinct CD spectrum with a single minimum at 198-200 nm.
While it may be tempting to attempt to study conformational changes that occur
on lipid binding using circular dichroism spectroscopy, lipid binding in micelles
using TAG, DAG, or cholesterol is substoichiometric, meaning that not all purified
proteins are bound to a ligand (
Gross et al., 2011; Radhakrishnan, Sun, Kwon,
Brown, & Goldstein, 2004
). This means that of the population of proteins in an ex-
periment, only a small minority will be bound to ligand at any one time, thus exceed-
ing the sensitivity of the circular dichroism method in detecting a conformational
change, as it depends on a statistical average.
10
5
0
-
5
-
10
190
200
210
220
230
240
250
260
Wavelength (nm)
FIGURE 11.3
Circular dichroism spectroscopy of FIT2-His
6
-StrepII demonstrates secondary structure.
Spectra of purified FIT2-His
6
-StrepII at a concentration of 3 mM in Buffer C were collected on
a Jasco J-815 CD spectrophotometer using a 2 mm path length cuvette at room temperature.
The average of six spectra is shown.
Adapted from
Gross et al. (2011)
.
