1. CHIRALITY IN BIOMOLECULES SYMMETRY AND ASYMMETRY IN NATURE
CIRCULAR DICHROISM AND O.R.D

3. M. Escher

4. Amino acids and sugars are chiral
Peptides, proteins, nucleotides, nucleic acids, sugars, polysaccarides and glycerophospholipid (from glycerol, which is pro-chiral) are intrinsecally chiral due to their primary structure.

5. Chirality criteria OPERATIONAL CRITERIUM:
Operationally:
Build (or imagine) the mirror image of the object.
If the object and its mirror image are superimposeable, then the object is not chiral.
Theoretically (Group Theory applied to chemical compounds):
Chirality derives from the lack of symmetry elements.
Axis of rotation are possible in chiral objects. Other symmetry elements (such as plane of symmetry, inversion center and axis of roto-reflection are not possible)
OPERATIONAL CRITERIUM:
Chiral objects are not superimposeable to their mirror images.

6. Chirality
aminoacid
nucleic acid
sugar

7. STEREO- ISOMERS, OPTICAL ISOMERS,
R----R S----S
R----S S----R
R---R---R S S S
R---S---S R---R---S
ENANTIOMERS R-
R---R R----S
R---R---R R—S---R
S---S---R S---R---S
DIASTEREOMERS

8. Levels of Protein Stucture
aminio acid
peptide bond
+H3N
COO-
a helix
b sheet
hydrogen bond
a. Primary structure
b. Secondary structure
c.Tertiary structure
d. Quaternary structure
disulfide bond

9. 5' RNA 1 2 O–PO–CH2 O U OH O–PO–CH2 O OH C OH O–PO–CH2 O G OH O–PO–CH2

10. Secondary structure chirality
protein a-helix (right-handed)
double stranded DNA helix, B-form (right-handed)

11. Helices and chirality
Note that the handness is retained when the helices are turned upside down.

12. Enantiomers and DiasteroisomersL L L L L L L L L
right-handed a-helix
left-handed a-helix
diastereoisomers
enantiomers
enantiomers

13. TAKE A PEPTIDE WITH 10 AMINO ACIDS
ASSUME THAT BOTH D AND L FORMS
ARE ALLOWED
HOW MANY STEREOISOMERS ARE POSSIBLE?
AND FOR A CHAIN WITH 50 AMINO ACIDS?

14. FOR A CHAIN WITH 50 racemic AMINO ACIDS,
THE POSSIBLE NUMBER IS
250 Many many billions......
What is the number when only one form is allowed?
This number is : ONE!!

15. } L D Asymmetry as a powerful ordering factor Hormone with 10 residuesNH 3 +
COO - R H L D
Asymmetry as a powerful ordering factor
Hormone with 10 residues
N = 2 x 2 x 2 x 10
= 10
if L and D isomers had the same probability.
But since only L-isomers are allowed, N = 1
For a chain with 50 residues ( Insulin ... )
N = 2 50 15
1 !
only L-residues
L - Asp - L - PheOMe Aspartame (sweet)
L D
D L
D D }
bitter

16. Macroscopic systems are highly symmetric

18. Plane-polarized light

19. Classification of Polarization
Light in the form of a plane wave in space is said to be linearly polarized. Light is a transverse electromagnetic wave, but natural light is generally unpolarized, all planes of propagation being equally probable. If light is composed of two plane waves of equal amplitude by differing in phase by 90°, then the light is said to be circularly polarized. If two plane waves of differing amplitude are related in phase by 90°, or if the relative phase is other than 90° then the light is said to be elliptically polarized.
Circularly polarized light may be produced by passing linearly polarized light through a quarter-wave plate at an angle of 45° to the optic axis of the plate.

20. a
specific rotation
d in dm; c in g/mL
molar rotation

21. Left- and right-handed polarized light
Linearly polarized light can be seen as composed by right- and left-handed circularly polarized light.
A chiral compound interacts with these two kinds of light in a different manner.
As consequence, the emerging light carries the information (ellipticity) about the chirality of the system.

22. Optical Rotatory Dispersion & Circular Dichroism
Interaction between E and
molecules (electrons)
has two effects on E
reducing the velocity
of propagation
decreasing the amplitude
of E
circular dichroism
(elliptically polarized light results)
molar ellipticity deg cm2 dmol-1

23. Absorption spectrum e
Positive Cotton Effect
Negative Cotton Effect

24. CD bands can be positive or negative
Absorption
(e)
Differential Absorption
(eL- eR)
CD band can have a positive or negative sign because is related to the difference:
De and molar ellipticities are related by the relation:

25. a-Helix
Myoglobin

26. b-Sheet

27. CD of proteins

28. CD spectra of secondary structure models. Reference CD spectra
The CD spectrum in the region of the amide bond absorption is a sensitive probe to study the secondary structure of a protein.

29. Evaluation of the secondary structure
0.683
0.047
0.270
Generally the fit is done with four or five basis spectra: a-helix, b-sheet, b-turns, random coil, and eventually including aromatic contribution

30. pH-induced denaturation of myoglobin
[q] pH
2.6
3.8
5.0
6.2

31. CD in the near-UV region (aromatic residues)
Native
Denaturated
[q]
[q]
10 000
100
100
200
220 nm
240
280
320 nm

32. CD of nucleic acids
Compacted DNA (psi form) in lecithin reverse micelles
w0 = 5
w0 = 7
w0 = 10
w0 = 13
water phase

33.  
Prism Polarizer

34. The most commonly used units are mean residue ellipticity,  (degree·cm2/dmol), and the difference in molar extinction coefficients called the molar circular dichroism, εL-εR =Δε (liter/mol·cm).
The molar ellipticity [] is related to the difference in extinction coefficients by
[] = 3298 Δε.

35. Typical Initial Concentrations
Protein Concentration: 0.5 mg/ml
Cell Path Length: 0.5 mm
Stabilizers (Metal ions, etc.): minimum
Buffer Concentration : 5 mM or as low as possible while maintaining protein stability
Contaminants: Unfolded protein, peptides, particulate matter (scattering particles)