2/25/2016

CD (Circular Dichroism)
Spectroscopy

• Circular dichroism (CD) is the differential

absorption of left- and right-handed
circularly polarized light.
• This phenomenon is exhibited in the
absorption bands of an optically active
molecule. CD can be used to help
determine the structure of macromolecules
(including the secondary structure of
protein and the handedness of DNA).

1
 2/25/2016

• A CD spectrometer is an instrument that

records this phenomenon as a function of
wavelength.
• Modern instruments, however, can
generally also record CD as a function of
temperature or chemical environment, at
several wavelengths.

CD and Protein

CD measures interactions of polarized light

with chiral protein components that are formed
into different types of 2°structure (asymmetric),
and with other chromophores (ligands or
prosthetic groups or aromatic R groups) that are
asymmetrically bound.

2
 2/25/2016

Light
Unpolarized light consists of waves vibrating in all planes
perpendicular to the direction of travel.

Plane polarized light has waves vibrating in a single

plane. In plane polarized light, the varying electric field of
the radiation has a fixed orientation.

In circularly polarized light, the direction of polarization

rotates with the frequency of the radiation. If you observe a
circularly polarized beam of light coming toward you, the
electric field can be rotating in either a clockwise direction
(right circularly polarized light) or a counterclockwise
direction (left circularly polarized light).

Polarization of light. Top, plane polarized light; bottom, circularly polarized light.

3
 2/25/2016

• Since circularly polarized light itself is "chiral", it

interacts differently with chiral molecules. That is,
the two types of circularly polarized light are
absorbed to different extents.

• In a CD experiment, equal amounts of left and

right circularly polarized light of a selected
wavelength are alternately radiated into a (chiral)
sample. One of the two polarizations is absorbed
more than the other one, and this wavelength-
dependent difference of absorption is measured,
yielding the CD spectrum of the sample.

• Asymmetric molecules and components

of molecules (e.g. D- and L-amino acids;
right- and left-handed protein helices; etc.)
preferentially absorb either left or right
circularly polarized light !

4
 2/25/2016

Delta absorbance

∆A (Delta Absorbance) is the difference between absorbance of

left circularly polarized (LCP) and right circularly polarized (RCP)
light (this is what is usually measured).

∆A is a function of wavelength, so for a measurement to be

meaningful the wavelength it was performed at must be known.

Since ∆A can be either positive or negative, a CD spectrum is

unlike a normal absorption spectrum.

Molar circular dichroism

• εL and εR are the molar extinction

coefficients for LCP and RCP light,
• C is the molar concentration
• l is the path length in centimeters (cm).

5
 2/25/2016

• Then ∆є is the molar circular dichroism.

This intrinsic property is what is usually
meant by the circular dichroism of the
substance.

Extrinsic effects on circular dichroism

• In many practical applications, the measured CD

is not simply an intrinsic property of the
molecule, but rather depends on the molecular
conformation. CD may also be a function of
temperature, concentration, and the chemical
environment, including solvents.
• In this case the reported CD value must also
specify these other relevant factors in order to
be meaningful.

6
 2/25/2016

Application to biological
molecules
• Circular dichroism is exhibited by biological
molecules, because of their dextrorotary and
levorotary components.
• Even more important is that a secondary
structure will also impart a distinct CD to its
respective molecules. Therefore, the alpha helix
of proteins and the double helix of nucleic acids
have CD spectral signatures representative of
their structures.

• The far-UV CD spectrum of proteins can

reveal important characteristics of their
secondary structure. CD spectra can be
readily used to estimate the fraction of a
molecule that is in the alpha-helix
conformation, the beta-sheet conformation
or some other (e.g. random coil)
conformation.

7
 2/25/2016

Circular dichroism spectra for polypeptides in various conformations.

• These assignments place important

constraints on the possible secondary
conformations that the protein can be in.
CD is a valuable tool, especially for
showing changes in conformation. It can,
for instance, be used to study how the
secondary structure of a molecule
changes as a function of temperature or of
the concentration of denaturing agents,
e.g. Guanidium hydrochloride or Urea.

8
 2/25/2016

• The near-UV CD spectrum (>250 nm) of

proteins provides information on the
tertiary structure. The signals obtained in
the 260-300 nm region are due to the
absorption, dipole orientation and the
nature of the surrounding environment of
the phenylalanine, tyrosine, cysteine (or S-
S disulfide bridges) and tryptophanamino
acids.

wavelength region for CD signal = same as absorbance

for that chromophore:

~185-240 nm ("far uv") for 2°and 3°structure (peptide

bond absorbance, aromatic R groups, disulfide bonds)

~260-290 nm ("near uv") for 3°structure (aromatic R groups)

9
 2/25/2016

• Unlike in far-UV CD, the near-UV CD

spectrum cannot be assigned to any
particular 3D structure. Rather, near-UV
CD spectra provide structural information
on the nature of the prosthetic groups in
proteins, e.g., the heme groups in
hemoglobin and cytochrome c.

• Visible CD spectroscopy is a very powerful

technique to study metal–protein
interactions, exp: Cu2+ and Ni2+ square-
planar complexes involving histidine .
Optical activity in transition metal ion
complexes have been attributed to
configurational and conformational effects.

10
 2/25/2016

• CD gives less specific structural information than

X-ray crystallography and NMR spectroscopy,
which both give atomic resolution data.
• However, CD spectroscopy is a quick method
that does not require large amounts of proteins
or extensive data processing. Thus CD can be
used to survey a large number of solvent
conditions, varying temperature, pH, salinity, and
the presence of various cofactors.

CD spectroscopy is particularly good for:

• determining whether a protein is folded, and if so characterizing its
secondary structure, tertiary structure, and the structural family to
which it belongs
• comparing the structures of a protein obtained from different sources
(e.g. species or expression systems) or comparing structures for
different mutants of the same protein
• studying the conformational stability of a protein under stress –
thermal stability, pH stability, and stability to denaturants -- and how
this stability is altered by buffer composition or addition of stabilizers
and excipients
• If there are any conformational changes, this will result in a spectrum
which will differ from the sum of the individual components. Small
conformational changes have been seen, for example, upon formation
of several different receptor/ligand complexes

11
 2/25/2016

Example of CD Application

30 40

25 35

30
20
25
CD in mdeg

15
CD in mdeg

20

10 15

10
5
5
0
0

-5 -5

-10
-10
-15
240 260 280 300 320 240 260 280 300 320

wavelength in nm wavelength in nm

Figure: CD spectra of BPN (left) and BPI2 (right) with the same conditions
(pure DNA spectra with added drug spectra black and complex
spectra red)

12

BRIEF SUMMARY OF METHODS FOR PROTEINS
TYPE PROTEIN COMPONENTS
UV-VIS spectroscopy
• peptide bonds (~220 nm)
• aromatic residues [esp. W
a) absorbance
(280 nm), (Y)]
• some ligands & prosthetic
groups
• W [λmax,ex=280m,
b) fluorescence λmax,em=~340nm]
• some ligands & prosthetic
groups
• secondary structure (180-
240nm) (peptide bonds)
c) circular dichroism (CD) • tertiary structure
(environment of aromatic
R groups)(260-300nm)
• ligands&prosthetic groups
2/25/2016
USES
• determine protein
concentration
• conformational changes
• ligand binding
• conformational changes
• ligand binding
• 2o structure (amount &
type)(far uv)
• conformational changes
(2o & 3o structural
changes)
• ligand binding
13