Circular Dichroism
Part II. CD spectra of Nucleocide
Electron Molecular Energy
Chromophores of Nucleic Acid
• p  p* transitions begin about 300 nm
• n  p* buried under p  p* transitions
The intensity of the CD is low because it is a secondary
effect of the asymmetric sugar inducing a CD in the
chomophoric, but symmetric base. 
Chromophores of Nucleic Acid
Absorption Spectra
CD Spectra
Base Stacking & CD spectra of
Nucleic Acid
• The benzene-like p-electron systems of the bases make them
hydrophobic, so the bases tend to stack in hydrogen bonding
solvents to minimize the p-electron surface area exposed to the
solvent.
• The NH, NH2 and CO groups are hydrophilic, so the edges of
the bases will interact well with hydrogen bonding solvents
• For nucleic acids the hydrophobic planes, the hydrophilic edges
and charge-charge interactions cause the bases to stack and the
polymers to assume a helical structure.
• The electronic transitions of the chromophoric bases are in close
proximity, and can interact to give CD spectra of high intensity.
Polymorphic properties of
Nucleic Acid
Polymorphism of nucleic acid in secondary structure
•Number of base pairs per turn
•Inclination of the base with respect to the helix axis
•The distance of the bases from the helix axis
•The rise per base-pair
•Handedness of the helix
CD can measure the change in secondary structure as a
function of solvent conditions.
From monomer to
polymer
Formation of helical
structure is a super
asymmetry that gives rise
to degenerate interactions
between chromophoric
bases and results in
intense CD spectra
CD of single stranded oilgo(rA) in
aqueous solution at pH 7
Polymer
dimer
monomer
Base-stacked helices in aqueous solution
Spectra is Composition dependent
CD of single stranded poly(rA)
CD of single stranded poly(rC)
CD of double-stranded
DNA and RNA
CD vs. Absorption
Native DNA
Denatured Native DNA
•CD occurs only where normal
absorption occurs
•CD is more complicated
revealing bands that not
separated in the normal
absorption spectrum
Average spectrum for the four
component deoxynucleotides
Structure of DNA
A-DNA
B-DNA
Z-DNA
Discovery of Z-form DNA
Pohl and Jovin (1972 JMB, 67,p375 ) were the first to observe the
left-handed Z-form of poly(dGC)-poly(dGC), and they did this by using
circular dichroism spectroscopy.
The Z-form DNA
•negative band at 290
•positive band at 260 nm.
•crossover about 185 nm
Z-form is not the mirror
image of the B-form, the
blue shift of the 200 nm of
the B-form to 185 nm in
the Z-form appears to be
the trademark of the B to Z
transition.
A-DNA
B-DNA
Z-DNA
DNA Secondary Structure &
CD Spectra
A DNA
260 nm positive
210 nm intense negative
190 nm intense positive
A-DNA
B DNA
275 nm positive
240 nm negative
258 nm crossover
B-DNA
The CD of E. coli DNA in
various structure
A-DNA
10.4 B-DNA
10.2 B-DNA
Sprecher et.al. Biopolymer 17,1009
Solvent Effect on DNA Structure I
Calf thymus DNA
10.4 base pair B-form
0% methanol
25% methanol
50% methanol
65% methanol
75% methanol
95% methanol
10.2 base pair B-form
Solvent Effect on
DNA Structure II
90% methanol
Titration with ethanol causes
the same changes as with
methanol in CD up to 65%.
Adding more ethanol causes
a change to A form
75% methanol
70% methanol
65% methanol
P Form DNA
P-DNA
(95%
methanol/5
% buffer)
330C
P-DNA (47.5%
methanol/5% buffer/4.5%
ethanol) 80C
B-DNA
P-DNA
10.2 B-DNA (95% methanol/5% 80C)
Temperature Effect
on DNA
58.30 C
48.20 C
The CD of poly(dA) poly(dT)
as a function of temperature
44.70 C
38.80 C
CD is sensitive to the change in
conformation when DNA melts
with increasing temperature
10 C
Triplex Nucleic Acid
177
Triplex
190
Poly(dA dT dT)
260
210
250
280
Duplex
Poly(dA dT)
With Mg+2
Intra-molecular triplex
Without Mg+2
With Mg+2
Intra-molecular triplex
Influence of the
temperature on
the parallel triplex
30C
triplex
260nm
280nm
660C duplex
930C
denature
Circular Dichroism
Part III. CD spectra of Protein
Amide Chromphore
• n  p* centered around 220 nm
• p  p* centered around 190 nm
n -> p* involves non-bonding electrons of O of
the carbonyl;
p -> p* involves the p-electrons of the carbonyl
Random coil
positive at 212 nm (p->p*)
negative at 195 nm (n->p*)
b -Sheet
negative at 218 nm (p->p*)
positive at 196 nm (n->p*)
a-helix
positive (p->p*)perpendicular at 192
nm
negative (p->p*)parallel at 209 nm
negative at 222 nm is red shifted
(n->p*)
Secondary Structure Determination
a , b , RC

i
f i Si ( )
TFE Induced Helix
Sodefrin&S10K-A3之CD測量圖
Helix Content = 100×(〔θ〕222／max〔θ〕222)
max〔θ〕
222=-40,000[1－(2.5/n)]，
n=胺基酸之殘基數
Behrouz Forood et.al Proc.Natl.Acad.Sci.USA Vol.90,pp.838～
842,February 1993
Applications of CD in Structural Biology
•Determination of secondary structure of proteins that
cannot be crystallised
•Investigation of the effect of e.g. drug binding on
protein secondary structure
•Dynamic processes, e.g. protein folding
•Studies of the effects of environment on protein
structure
•Secondary structure and super-secondary structure of
membrane proteins
•Study of ligand-induced conformational changes
•Carbohydrate conformation
•Investigations of protein-protein and protein-nucleic
acid interactions
Software for the Analysis of
Circular Dichroism Data
Tools for analyzing circular dichroism data :
• LINCOMB and MLR( The method of least squares)
• CONTIN (The ridge regression procedure of Provencher and
Glöckner)
•
VARSLC (The Variable Selection Method of Johnson and
Coworkers )
•
SELCON (The Self-Consistent Method of Sreerama and
Woody )
•
•
•
K2D.(A neural net analysis program of Andrade et al)
CCA (The convex constraint algorithm of Fasman and coworkers )
SVD (Singular Value Decomposition ).
http://lamar.colostate.edu/~sreeram/CDPro/