# Lecture 3

```Physical Methods in Biochemistry:
BIOC-522
Polarization of Light; Circular Dichroism (CD)
and its Application in Proteins and Nucleic Acids
Balachandra Hegde
Zilkha Neurogenetic Institute, Room 121
Tel. (323) 442-0106
e-mail: [email protected]
08-30-11
Introduction:
Electromagnetic Wave
Unpolarized light
Unpolarized light consists of electric/magnetic vectors vibrating in
many directions perpendicular to the direction propagation.
Polarization
Plane (Linear) Polarization
If the vector of the electric field oscillates along a straight line then the
waves are called plane-polarized or linearly polarized waves.
http://www.enzim.hu/~szia/cddemo/edemo2.htm
Addition (superposition) of plane polarized waves
• Two waves add according to vector addition rules.
•The resulting waves depend on the amplitude and phase of
two waves.

If two sine waves have the same
frequency and occur at the same
time, they are said to be in phase
If the two waves occur at different
times, they are said to be out of
phase
Adding two “in phase” plane polarized waves
generate a “plane” polarized wave
http://www.enzim.hu/~szia/cddemo/edemo2.htm
Circular Polarization
Adding two “out of phase” plane polarized waves generate a
“circularly” polarized wave
When two waves plane-polarized in two perpendicular planes
meet out of phase then the resulting wave is circularly polarized.
Phase
difference
90o
-
90o
Right circularly
Polarized light
Left circularly
Polarized light
http://www.enzim.hu/~szia/cddemo/ede
mo2.htm
Plane polarized light is the sum of left and right circularly
polarized light.
Any plane polarized light wave can be obtained as a
superposition of a left and a right circularly polarized light
wave, whose amplitude is identical
In other words plane polarized light has two circularly polarized
components.
http://www.enzim.hu/~szia/cddemo/edemo2.htm
Elliptically -Polarized Light
When plane polarized light passes through a solution containing an optically
active substance (Chiral) the left and right circularly polarized components of
the plane-polarized light are absorbed by different amounts.
When these components are recombined they appear as elliptically polarized
light.
http://www.enzim.hu/~szia/cddemo/edemo2.htm
Summary
•Plane polarization: If the vector of the electric
field oscillates along a straight line.
•Addition of two waves
•Adding two in phase wave produce linearly polarized light
•Adding two out of phase waves produce circularly polarized
light.
Plane polarized wave has two circularly polarized
components, left- and right.
Elliptically polarized light: In an optically active
medium right- and left- circularly polarized light are
absorbed by different amount.
Plane (Linear) Polarization
Circular Polarization
Circular Dichroism (CD)
• When plane polarized light passes through a solution
containing an optically active substance (Chiral) the left
circularly polarized light absorbed to a different extent than
right circularly polarized light.
• The result is an elliptically polarized light.
• The origin is molecular asymmetry.
Semimajor axis
a
b
Semiminor axis
 = tan-1 b/a
  b/a, when  is small.
CD can be characterized by the ratio of the semiminor and semimajor axes,
which is the tangent of an angle , called ellipticity. The ellipticity, , is the
angle of polarization and is measured in degrees, (deg) or millidegrees,
(mdeg).
CD is also defined as the difference between the absorption of
Left Circularly Polarized light (LCP) and Right Circularly Polarized light
From Beer‟s law absorption of LCP is
(RCP).
I
AL = log10(ILo/IL) = l c l
ILo and Il are intensities of incident and emergent LCP light resply.
c - molar concentration of the medium of path length l and
L - is the extinction coefficient of LCP.
Similarly for RCP
AR = log10(IRo/IR) = R c l
A = AR – AL
= R c l - L c l = (R - L ) cl
=  c l
 is called decadic molar CD
It is common practice to convert measured quantity (AL – AR) to ellipticity using the
equation
 = 2.303 (AL – AR) 180 / 4
degrees
To compare results from different samples, it is necessary to compute optical activity
on molar or residue basis
Mean residue molar ellipticity
[]MRE = millidegree / cM . lmm . nresidues [deg cm2 dmol-1]
[]MRE = [] / nresidues,
[] is molar ellipticity
[] = millidegree / cM . lmm = mdeg/M.mm = mdeg/mol L-1 . mm
= 10-3 deg / mol . 103 m-3 .10-3 m (since 1L = 103 cm3
=
10-3 m3 )
= deg / 103 .mol . m-2 = deg / 103 .mol . 10-4 cm-2 = deg / 10-1 .mol .cm-2
[] = deg cm2 dmol-1
In practice the formula used is (ellipticity per residue per molar)
[] = obs
[
1
] deg cm
No. of residues X Molar concentration X path length in mm
2
dmol-1
Instrumentation
• JASCO - 810 spectropolarimeter
• Single position peltier for temperature regulation and temperature
scanning
• Biologic stopped-flow system for rapid kinetics
Commercial instruments measure CD by using a modulation
technique to measure the generally very small A
Block Diagram of the JASCO J-810 Spectropolarimeter
Xenon lamp
Crystal prisms which produce linearly polarized light
Modulator
Sample
Atmosphere in the light source unit, monochromator unit, and sample
chamber is displaced using dry N2 gas as Xenon lamp produces Ozone
which absorbs UV.
Some practical aspect
Buffers - use lowest concentration possoble. Typically 10 mM. Filter buffers through syringe
filters prior to use.
use:
Phosphate buffers
HEPES buffers
Ammonium Acetate buffers
avoid:
Tris buffers
Glutamate or Glycine buffers (or anything chiral)
Salts - Try to use as little as possible. KCl is better than NaCl. NaF is suggested if very low
wavelengths are needed.
Peptide/protein concentration - literature says 0.5 mg/ml is good starting point. Signal
intensity will depend on polypeptide length. For a 20 residue peptide 20 μM gives good signal. For
300 residue protein (like annexin) 1 μM is sufficient.
Sample volume - minimum of 175 uL for the 1 mm cell. We also have a demountable flat cell
which may take less volume.
(High concentrations of DTT, histidine, or imidazole, cannot be used in the far-uv region.)
Chirality
• A property of most biological molecules is molecular
asymmetry, or chirality.
• A molecule is chiral if it is non-superimposible on its mirror
image.
The left hand is a non
super imposable mirror
image of the right hand.
All molecules with a chiral center are also optically active- they rotate
plane-polarized light and have different absorption of left- and rightcircularly polarized light.
Amino acids
• In all standard amino acids (except glycine), the - carbon is bonded to
four different groups: carboxyl group, amino group , R-group and
hydrogen atom
• These four different groups can occupy two different spatial arrangements
that are non superimposible mirror images of each other, called
enantiomers or optical isomers.
D-isomer
Dextrorotatory
Rotates plane of polarized
light to the right
Thus the - carbon atom is a chiral center.
L-isomer
Levorotatory
Rotates plane of polarized light
to the left
Peptide and protein
Carboxyl group
Amino group
Far-UV CD is used for determining protein structure.
Amide bond electrons absorb in this energy range:
n -&gt; *, promotion of an electron from a 'non-bonding' (lone-pair) n orbital to
an 'antibonding' π orbital: centered around 220 nm
 -&gt;  *, promotion of an electron from a 'bonding' π orbital to an 'antibonding'
π orbital: centered around 190 nm
The intensity and energy of these transitions depends on the angles the
peptide bond assumes (,  angles) and therefore on the secondary structure
of the protein.
Determination of Protein Secondary Structure
- helix: (pH 10.8, 25oC)
-ve band at about 222 nm
-ve and +ve couplet at about 208 and 190 nm
The magnitude of the –ve 222 nm band is a good measure of  helix
content in a peptide or protein.
There is a fairly linear relationship with [] = 0 correspond to 0%
 - helix and [] = -36 &plusmn; 3 x 103 corresponding to 100%
- sheet: (pH 10.8, 52oC)
-ve band at about 215 nm
+ ve band at 198 nm
Positions and intensities of these two
bands vary from sample to sample
Wavelength (nm)
CD spectra of poly-L-Lysine
Random coil: (pH 6, 25oC)
Intense –ve band around 198 nm
+ve band around 218 nm
190
198
 - helix
 - sheet
215
208
222
Wavelength (nm)
Random coil
218
198
http://mach7.bluehill.com/proteinc/cd/cdspec.html
Absorption of Energy
 helix
 sheet
Random
coil
-*
positive
190-195
nm
60 - 80 x 103
deg cm2 dmol-1
-*
negative
208 nm
-36 &plusmn; 3 x 103
n-*
negative
222 nm
-36 &plusmn; 3 x 103
-*
positive
195-200
30 - 50 x 103
n-*
negative
215-220
-10 - (-20) x 103
-*
negative
198-200
-20 x 103
n-*
positive
220
small
Sheet
deg
d
deg
d
RC
Wavelength
Wavelength
Analysis of the secondary structure:
[(l)] = fS(l) + fS(l) + fRCSRC(l),
where S(l), S(l), and SRC(l) are derived from poly-L-lysine basis spectra.
Three unknowns f, f ,fRC can be estimated by
solving minimum 3 simultaneous equations by
least square method.
B
50
45
40
35
30
25
20
15
10
5
0
-5
-10
-15
-20
-25
200
210
220
230
240
250
260
Wavelength (nm)
This method is usually accurate to within 10% for  -helix content.
There are problems with this assumption:
• The basis spectra for each structural class is not easy to obtain. Easy to a obtain helical basis set
but not so easy for β-sheets, turns or random classes.
• A CD spectrum cannot always be formed from the linear combination of basis spectra. Must take
into account aromatic contributions, contributions from tertiary interactions etc.
• Contribution of helix to the spectrum depends on the length of the chain.
Therefore other methods have been developed which are better:
• Ridge Rigression
• Singular Value Decomposition
• Convex Constraint Analysis
• Principle Component Factor Analysis
• Backpropagation Neural Networks
Methods do not rely on a basis set of
„pure‟ spectra.
Programs:
DICHROPROT - from http://dichroprot-pbil.ibcp.fr/
• Least Squares
• Variable Selection Method - SVD
• Self Consistent Method - SVD
• K2D Neural Network Method
• CONTIN Method - ridge regression
• Based on Θ @ 220 nm - for peptide helicity
CDPRO - from http://lamar.colostate.edu/~sreeram/CDPro/
• SELCON3 - self consistent method, SVD
• CDSSTR - variable selection, SVD
• CONTIN - ridge regression
CDNN - neural network based @ http://bioinformatik.biochemtech.unihalle.de/cd_spec/index.html
Demonstrating Comparability of Conformation
•Often it is necessary to demonstrate that different lots of a protein
have equivalent conformations.
•far-uv spectra show that the recombinant form of an enzyme
clearly does not have the same secondary structure as the natural
protein (i.e. the recombinant protein is not properly folded).
CD of Nucleic Acids
Forms of DNA
36 base pair structure
A–
form
B–
form
Z–
form
Helical
sense
Right
handed
Right
handed
Left
handed
Diameter
~ 26 &Aring;
~ 20 &Aring;
~ 18 &Aring;
Base pair
per helical
turn
11
10.5
12
Helix rise
per base
pair
2.6 &Aring;
3.4 &Aring;
3.7 &Aring;
•CD is the characteristic of the conformation of the nucleic acid
•It depends on base composition, because each base has different transition
dipoles.
• The +ve and -ve CD couplet about 280
and 240 nm and the intense +ve at about
190 nm are characteristics of the 10.4 bp
B-form
A-form in 80% TFE
10.4 bp B-form in
aqueous buffer at pH 7.0
• Collapse of the long wavelength 280 nm CD
band is the hallmark of of the 10.2 bp B-form
10.2 bp B-form in 6M NH4F,
aqueous buffer pH 7.0
CD of E. coli DNA in various secondary structures
•
The more intense +ve CD at 270 nm
coupled with a –ve CD at 210 nm, and
an extremely intense +ve CD at 185 nm,
are characteristic of the A-form.
CD can be used to monitor the conformational changes in nucleic acid as a
function of solvent.
1)
2)
3)
4)
5)
6)
0% methanol
25%
50%
65%
75%
95%
CD of calf thymus DNA in 0.005M standard sodium citrate as a
function of methanol conc: long wavelength portion of the CD
changes as methanol converts it from the 10.4 bp B-form to the 10.2
bp B-form
The first observation of Z-form DNA was by use of CD
The CD of poly d(GC) . Poly d(GC) as various secondary structure
B – form in aqueous buffer at pH 7
Z – form in 2M sodium perchlorate at pH 7
A – form in 80% TFE at pH 7
Blue shift of the 200 nm crossover of the B – form to about 185 nm in the
Z – form appears to be the trademark of the B to Z transition.
CD application
• Can be used to determine the secondary structure of
polypeptides and nucleic acids.
• Can be used to look at structural changes of polypeptides
and nucleic acids under various conditions - temperature, pH,
salt, kinetics.
• Can be used to judge the proper folding of polypeptides
obtained from different sources (recombinant vs. synthetic).
• Can be used to measure structural changes resulting from
protein-protein or protein-membrane interactions.
References:
Principles of Physical Biochemistry, 2nd edition
K. E. van Holde, P. Shing Ho.
2. Biophysical Chemistry Part II: Techniques for the study of
biological structure and function,
Cantor and Schimmel.
3. Circular Dichroism and the conformational analysis of biomolecules,
Edited by Gerald D. Fasman.
4. Some figures and animations are from internet.
1.
Some useful web sites.
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
http://www.enzim.hu/~szia/cddemo/edemo2.htm
```