Rotation of Plane-polarized Light by
an Optically Active Sample
Structural Analysis of Protein
Structure
Circular Dicroism
Fluorescence
Methods for Secondary Structural Analysis
• A number of experimental techniques can selectively
examine certain general aspects of macromolecular
structure with relatively little investment of time and
sample.
• Reasonable estimates of protein secondary structure
content and structure change can be determined
empirically through the use of
Circular dichroism (CD) spectroscopy
Fluorescence spectroscopy
Nuclear Magnetic Resonance (NMR) spectroscopy
FT-infrared spectroscopy
Circular Dichroism
• Circular dichroism (CD) spectroscopy is a form of light
absorption spectroscopy that measures the difference in
absorbance of right- and left-circularly polarized light (rather
than the commonly used absorbance of isotropic light) by a
substance.
• It is measured with a CD spectropolarimeter. The instrument
needs to be able to measure accurately in the far UV at
wavelengths down to 190 - 170 nm (170 - 260 nm).
• The difference in left and right handed absorbance A(l)- A(r)
is very small (usually in the range of 0.0001) corresponding to
an ellipticity of a few 1/100th of a degree.
• Pockels cell produces a beam that is alternately switched
between L and R. The beam then passes through the
sample to a photomultiplier. The detected signal can then
be processed as ΔA vs λ.
Instrumentation
• The most common instruments around are the
currently produced JASCO, JobinYvon, OLIS,
and AVIV models.
• We have the Jasco 710 and 810 models with
temperature controllers. The air cooled 150W
Xenon lamp does not necessitate water cooling.
• You still need to purge with ample nitrogen to get
to lower wavelengths (below 190 nm).
Typical Initial Concentrations
• Protein Concentration: 0.5 mg/ml (The protein concentration
needs to be adjusted to produce the best data).
• Cell Path Length: 0.5-1.0 mm. If absorption poses a problem,
cells with shorter path (0.1 mm) and a correspondingly increased
protein concentration and longer scan time can be employed.
• Stabilizers (Metal ions, etc.): minimum
• Buffer Concentration: 5 mM or as low as possible, while
maintaining protein stability. A typical buffer used in CD
experiments is 10 mM phosphate, although low concentrations
of Tris, perchlorate or borate is also acceptable.
• As a general rule of thumb, one requires that the total
absorbance of the cell, buffer, and protein be between 0.4 and
1.0 (theoretically, 0.87 is optimal).
• A spectra for secondary structure determination (260 - 178 nm)
will require 30-60 minutes to record (plus an equivalent amount
of time for a baseline as every CD spectrometer.
1
 Sample Preparation and Measurement
• Additives, buffers and stabilizing compounds: Any compound,
which absorbs in the region of interest, (250 - 190 nm) should be
avoided. A buffer or detergent, imidazole or other chemical should
not be used unless it can be shown that the compound in question
will not mask the protein signal.
• Protein solution: The protein solution should contain only those
chemicals necessary to maintain protein stability/solubility, and at
the lowest concentrations possible. The protein itself should be as
pure as possible, any additional protein will contribute to the CD
signal.
• Contaminants: Particulate matter (scattering particles), anything
that adds significant noise (or artificial signal contributions) to the
CD spectrum must be avoided. Filtering of the solutions (0.02 m
syringe filters) may improve signal to noise ratio.
• Data collection: Initial experiments are useful to establish the best
conditions for the "real" experiment. Cells of 0.5 - 1.0 mm path
length offer a good starting point.
CD Data Analysis
• The difference in absorption to be measured is very small.
The differential absorption is usually a few 1/100ths to a
few 1/10th of a percent, but it can be determined quite
accurately. The raw data plotted on the chart recorder
represent the ellipticity of the sample in radians, which can
be easily converted into degrees
(radians)
(degrees)
CD Data Analysis
• To be able to compare these ellipticity values we need to
convert into a normalized value. The unit most commonly
used in protein and peptide work is the mean molar
ellipticity per residue. We need to consider path length l,
concentration c, molecular weight M and the number of
residues.
in proper units (CD spectroscopists use decimol)
which finally reduces to
The values for mean molar ellipticity
per residue are usually in the 10,000's
CD Data Analysis
• The molar ellipticity [] is related to the difference in
extinction coefficients
[] = 3298 Δε.
• Here [] has the standard units of degrees cm2 dmol -1
• The molar ellipticity has the units degrees deciliters
mol-1 decimeter-1.
Circular Dichroism of Proteins
• It has been shown that CD spectra between 260 and
approximately 180 nm can be analyzed for the different
secondary structural types: alpha helix, parallel and anti-
parallel beta sheets, turns, and other.
• A number of excellent review articles are available
describing the technique and its application (Woody, 1985
and Johnson, 1990).
• Modern secondary structure determination by CD are
reported to achieve accuracies of 0.97 for helices, 0.75 for
beta sheet, 0.50 for turns, and 0.89 for other structure types
(Manavalan & Johnson, 1987).
CD Signal of Proteins
• For proteins we will be mainly concerned with absorption
in the ultraviolet region of the spectrum from the peptide
bonds (symmetric chromophores) and amino acid
sidechains in proteins.
• Protein chromophores can be divided into three classes: the
peptide bond, the amino acid sidechains, and any
prosthetic groups.
• The lowest energy transition in the peptide chromophore is
an n → p* transition observed at 210 - 220 nm with very
weak intensity (emax~100).
----p* p → p* ~`190 nm emax~7000
----n n → p* 208-210, 191-193 nm emax~100
----p
2
 Protein CD Signal
• The three aromatic side chains that occur in proteins (phenyl
group of Phe, phenolic group of Tyr, and indole group of
Trp) also have absorption bands in the ultraviolet spectrum.
However, in proteins, the contributions to the CD spectra in
the far UV (where secondary structural information is
located) is usually negligible. Aromatic residues, if
unusually abundant, can have significant effects on the CD
spectra in the region < 230 nm, complicating analysis.
• The disulfide group is an inherently asymmetric
chromophore as it prefers a gauche conformation with a
broad CD absorption around 250 nm.

Comparison of the
UV absorbance
(left) and the
Far UV CD Spectra of Proteins
circular dichroism
(right) of poly-L-
[] x10-3 degrees cm2 dmol -1

lysine in different
secondary structure
conformations as a
function of pH.

• The n → p* transition appears in the a-helical form of the

polymer as a small shoulder near 220 nm on the tail of a much
stronger absorption band centered at 190 nm. This intense band,
responsible for the majority of the peptide bond absorbance, is a
p → p* transition (emax ~ 7000).
• Using CD, these different transitions are more clearly evident.
Exciton splitting of the p → p* transition results in the negative
band at 208 and positive band at 192 nm.

CD Spectra of Proteins CD Spectra of

• Different secondary structures of peptide bonds have
different relative intensity of n → p* transitions, resulting
• Each of the three
basic secondary Protein
in different CD spectra at far UV region (180 - 260 nm). structures of a
• CD is very sensitive to the change in secondary structures polypeptide chain
of proteins. CD is commonly used in monitoring the (helix, sheet, coil)
conformational change of proteins. show a characteristic
CD spectrum. A
• The CD spectrum is additive. The amplitude of CD curve protein consisting of
is a measure of the degree of asymmetry. these elements should
• The helical content in peptides and proteins can be therefore display a
estimated using CD signal at 222 nm spectrum that can be
e222= 33,000 degrees cm2 dmol -1 res-1 deconvoluted into the
• Several curve fitting algorithms can be used to deconvolute three individual
relative secondary structures of proteins using the CD contributions.
spectra of proteins with known structures.

3
 CD Spectra Fit Conformational Change of CD2
c
• In a first approximation, a CD spectrum of a protein or
polypeptide can be treated as a sum of three components: 0

a-helical, b-sheet, and random coil contributions to the

6M GuHCl
spectrum.
• At each wavelength, the ellipticity (θ) of the spectrum will

[ ] (deg cm2 dmol )

-1
contain a linear combination of these components: -1000

25 ºC
(1)
-2000
• θT is the total measured ellipticity, θh the contribution from
helix, θs for sheet, θc for coil, and the corresponding χ the 85 ºC
fraction of this contribution.
-3000
200 210 220 230 240 250 260
Wavelength (nm)

CD2 Becomes Significantly Helical in TFE

CD Spectra Fit
• As we have three unknowns in this equation, a 5000

measurement at 3 points (different wavelengths) would

suffice to solve the problem for χ, the fraction of each 0
[] (deg cm2 dmol-1 res -1)

contribution to the total measured signal.

• We usually have many more data points available from our -5000
measurement (e.g., a whole CD spectrum, sampled at 1 nm 0% TFE
10% TFE
intervals from 190 to 250 nm). In this case, we can try to -1 10 4 17% TFE
minimize the total deviation between all data points and 19% TFE
30% TFE
calculated model values. This is done by a minimization of 4
80% TFE
-1.5 10
the sum of residuals squared (s.r.s.), which looks as
follows in our case : 4
-2 10
200 210 220 230 240 250 260

Wavelength (nm)

DICHROWEB Melting point measurement of EGFP-wt with its CD

spectra at different temperatures
http://www.cryst.bbk.ac.uk/cdweb/html/home.html

EGFP-wt Tm measurement Curve fitting of EGFP-wt

20 -1
A y = M1+M2/(1+ exp(-(M0-M3)/M... B
35 -2 Value Error
45
15 55 m1 -8.614 0.024757
65 -3 m2 7.2129 0.047864
CD (218 nm), mdeg

75 m3 65.718 0.11006
10 85
-4 m4 2.644 0.095431
95
CD, mdeg

Chisq 2.7395 NA
R 0.99849 NA
5 -5
B
-6
0
-7
Tm = 65.7 °C
-5
-8

-10 -9
200 210 220 230 240 250 260 0 20 40 60 80 100
Wavelength, nm Temperature, C

4
 Fluorescence measurement

Fluorescence spectrum of proteins Xe lamp

lex (nm) lem (nm) e (M-1 cm-1) Y

Trp (W) 280 348 5600 0.2
Tyr (Y) 274 303 1490 0.1
Phe (F) 257 282 240 0.04

Electronic mechanism of fluorescence Trp Fluorescence Emission Spectra of CD2

under Different Conditions
1st exited singlet state Triplet state
Intersystem crossing
• In a hydrophobic
environment (inside of
4 10 4

Trp
c
a folded protein), Trp
25ºC
emission occurs at
shorter wavelength.
Fluorescence intensity

4
3 10

Absorption fluorescence Phosphorescence

6M GuHCl When it is exposed to
hvex
4
2 10

hvem
85ºC
solvent, its emission is
vex > vem 1 10 4

very similar to that of

Vibration levels or the free Trp amino acid
lex < lem 0
300 320 340 360
Wavelength (nm)
380 400
(red shift occurs).
Ground singlet state

5
 Summary of fluorescence
• Fluorescence is the emission of radiation that occurs when a
molecule in an excited electronic state returns to the ground state.
• Application: Fluorescence has an important role in the structural
determinants of proteins, DNA or RNA, etc.
• Advantages:
– Small sample volumes (800μL – 3mL)
– Low concentration (0.1 – 5 M)
– Short experiment time (10-60 minute)
– Short data analysis time (5-30 minute)
– Recovery of sample
• Disadvantages:
– Large Stoke’s Shift
– Background fluorescence (Impurities in buffers and
autofluorescence in cells)
– Scattered light (problem with cloudy samples)