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

  M. Escher

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.

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.

Chirality aminoacid nucleic acid sugar

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

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

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

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

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

Enantiomers and Diasteroisomers L L L L L L L L L right-handed a-helix left-handed a-helix diastereoisomers enantiomers enantiomers

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?

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!!

} L D Asymmetry as a powerful ordering factor Hormone with 10 residues NH 3 + COO - R H L D Asymmetry as a powerful ordering factor Hormone with 10 residues N = 2 x 2 x 2 x 2 ................................. 2 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

Macroscopic systems are highly symmetric

Plane-polarized light

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.

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

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.

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

Absorption spectrum e Positive Cotton Effect Negative Cotton Effect

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:

a-Helix Myoglobin

b-Sheet

CD of proteins

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.

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

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

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

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

  Prism Polarizer

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.

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)