Best practice in CD
Nicholas C. Price
IBLS
University of Glasgow
Efraim Racker (1913-1991)
“Don’t waste clean
thoughts on dirty
enzymes”.
An example of poor CD data
A glycoprotein – CD
spectra recorded at
progressively higher
concentrations (e to a)
At high protein
concentrations,
the spectra
reflect the “HT
voltage artefact”.
An example of incorrect scaling
Stated to be typical
of a protein with
significant -helix.
However, the values
of [] are about 5
times too small.
Far UV CD of BSA at
pH 7.0 (a) and 4.2 (b).
A further example of incorrect scaling
Vis-near UV CD spectrum of ETF (Electron
Transferring Flavoprotein) (WT and mutant)
ETF CD spectrum (continued)
•According to the text, the molar concentrations
are expressed in terms of the flavin.
•In this case, we would need to use a protein
concentration of about 250 mg/ml in a cell of
pathlength 1 cm to obtain a maximum signal of 1
mdeg.
•Much more likely is that the molar
concentrations are expressed in terms of the
peptide bond unit (some 550-fold higher).
Main sections of talk
Stages of CD analysis
Preparation of sample
Acquisition of data
Presentation and analysis of data
The stages of CD analysis
•Preparation and characterisation of sample
•Acquisition of data
•Presentation and analysis of data
Each of these must be carefully checked!
Preparation and characterisation of sample
(1)
•Most protein samples are now produced by
heterologous over-expression.
•Is the protein pure and of the correct sequence;
have any tags been removed and have any posttranslational modifications occurred?
•Is the protein stable under the conditions
studied?
Preparation and characterisation of sample
(2)
How do we determine the concentration of
the sample?
Methods available include:•Coomassie Blue binding (Bradford assay)
•Bicinchoninic acid (BCA) assay
•UV absorption (280 nm or 205 and 280 nm)
•Amino acid analysis
Each has its merits but AAA is the gold
standard.
Preparation and characterisation of sample
(3)
•Make sure that the protein is stable in the
solvent used and that solvent absorbance is
not too high.
•Avoid high concentrations of Cl- ions,
carboxylate ions and imidazole, especially for
far UV CD studies.
•Always run a solvent blank and check the HT
voltage over the wavelength range.
•If necessary, adjust cell pathlength and
protein concentration to optimise conditions.
Effect of buffers on CD spectra (1)
1, 50 mM sodium phosphate, pH 7.4; 2, plus 150 mM
NaCl; 3, plus 150 mM imidazole; 4, plus 50 mM acetate
Effect of buffers on CD spectra (2)
Acquisition of data (1)
Is the spectropolarimeter regularly
maintained and calibrated?
Calibration of wavelength using (among
others):•Holmium oxide (279.4, 361.0 and 453.7 nm)
•Benzene vapour (241.7, 253.0 and 260.1 nm)
•Neodymium glass (586.0 nm)
Best to check calibration at a number of
wavelengths.
Acquisition of data (2)
Amplitude calibration can be achieved
using:•Camphor Sulphonic Acid (peaks at
290.5 nm and 192.5 nm)
•Pantolactone (peak at 219 nm)
•Tris(en)Co complex (peak at 490 nm)
Acquisition of data (3)
Is the cell properly cleaned and is its
pathlength known accurately?
Pathlengths can differ significantly from
nominal values for short pathlength cells
(<0.01 cm), and especially demountable
cells.
Acquisition of data (4)
Are the machine settings optimised?
•Bandwidth
•Time constant (response time, dwell
time)
•Scan rate
•Number of scans
Useful guidelines are:SR x TC < bandwidth < W/10
(where W is width at ½ height of spectral
feature).
Effect of machine settings on CD spectra
Solid line, 0.5 s, 50 nm/min; dotted line, 2 s, 50
nm/min; dashed line, 2 s, 100 nm/min.
Presentation and analysis of CD data
• CD data are presented in terms of either ellipticity
(degrees) or absorbance.
• In order to normalise data, they are referred to
molar concentrations of chromophore or
repeating unit.
• For far UV CD, the repeating unit is the peptide
bond.
• The mean residue weight (MRW) is calculated
from the molecular mass/(N – 1); N = number of
amino acids.
• For most proteins, MRW is around 110.
Units for CD data (1)
To calculate mean residue ellipticity ([]mrw )
[]mrw = (MRW x obs) deg. cm2.dmol-1
10 x d x c
Where
obs
d
c
= observed ellipticity in degrees
= pathlength in cm
= concentration in g/ml
Units for CD data (2)
If we know the molar concentration (m) of a
solution:[]molar = (100 x obs) deg.cm2.dmol-1
(d x m)
where d is the pathlength in cm.
Units for CD data (3)
For data in absorption units, it is usual to calculate
the molar extinction coefficient
= 
L– 
R
in units of M-1.cm-1
If a solution of molar concentration m in a cell of
pathlength d cm gives a difference in absorbance
(AL - AR) of Aobs, the value of is given by:= Aobs
M-1.cm-1
dxm
Units for CD data (4)
• Note the relationship
[]mrw = 3298 
• There is some debate about the correct choice of
molar units for near UV and visible CD – does it
refer to the MRW or to the intact protein? The
paper should state this clearly.
Analysis of CD data (1)
• There is a variety of algorithms for far UV CD
analysis, e.g. SELCON, VARSLC, CDSSTR,
CONTIN and K2d.
• These can differ in terms of reference sets of
proteins and wavelength ranges covered.
• The online server DICHROWEB (hosted at
Birkbeck College) allows easy data input and
analysis.
Analysis of CD data (2)
Important criteria for assessing the reliability of
analysis include:•Goodness of fit parameter NRMSD (should aim for
<0.1, or ideally <0.05)
•Difference (R) between X-ray and CD analysis
(should aim for <0.1)
•Comparison of calculated (reconstructed) and
experimental spectrum (systematic differences?)
•Consensus estimates of secondary structure
using different algorithms
References
• Jones, C. et al. (2004) NPL Report, ISSN 17440602
• Kelly, S.M. et al. (2005) Biochim. Biophys. Acta
1751, 119-139
• Miles, A.J. et al. (2003) Spectroscopy 17, 653-661
• Miles, A.J. et al (2005) Spectroscopy 19, 43-51
• Whitmore, L. and Wallace, B.A. (2004) Nucl. Acids
Res. 32, W668-W673