1

Polarization and Circular Dichroism - 2014 (Notes 17)

Since is vector, if fix molec. orient., E-field interact (absorb) with molecule differently when change E-orientation (polarization)

Transitions can be allowed for x,y,z orient in molecule

e.g. s px in H-atom, excite by Ex x, (but not orient !)

* in ethylene, C2H4, polarize along C=C bond – gas or solution—no impact / orientations average out – solid orient molecule - crystal-small molecule-fixed xyz

Alternative to crystal – dissolve in oriented material a) liquid crystal – locally orient. – long axis of inserted molec.

favor orientation –e.g. polyene, retinal, cholesterol

b) lipid membrane – composed of charged head groups and alkyl tails, bilayer form: -- self-assembles in layers – alkyl interior favor hydrophobic

-- packing favors orient. tails

Protein helices (hydrophobic) can insert

in membrane, orient surface, alkyl tails - e.g. trans membrane protein/peptide –antibiotics (leak, ~1), bundle for channels (signals, neural, 2) Can also make channels with sheets (3)

2

–Alternate: alkyl tail, hydrophilic res.– bind surface charges (PO2

-)

- amphipathic helices (+/- sides) can lay on surface, hydrophobic penetrate, charges out

or form assemblies-helical clusters, insert charged side in

c) Flow - long molec. orient flow Works well for DNA, fibers, etc.

d) surfaces, and reflection, provide alternate - sense polarization, s&p

Useful if chromophore – absorbing species – has different absorbance with one polarization – called dichroism (linear)

– can use for analysis of orientation

ex. IR studies easier to understand, C=O str. (amide I)

dipole along C=O bond, in helix,

to axis, in sheet

ex.-lactoglobulin interacts with

membrane and converts from -

sheet to -helix. ATR polarization difference spectra senses lipid & amide

3

High density lipo-protein (HDL) can make a discoid with lipid Polarized IR can tell orientation of the helices, in the plane of the lipid bilayer, so can be placed as belt around discoid

Out of

plane,

In plane,

4

Circular Polarization

Phase shift - 2 E-M waves

displaced by /4 along z combine rotates E as propagate (trace helix, L - R)

https://www.youtube.com/watch?v=Fu-aYnRkUgg

Make by mix two beams, shift phase in birefringent crystal

Circular Polarization Right or Left use birefringent plate with nx≠ny - retard (slow) Ex or Ey one polarization relative to other, tune: adjust path or stress Molecule sees both linear polarizations (Ex + Ey) oscillate

but due to the rotation between them at (circularity) has different selection rules – Chiral molec, AL ≠ AR

Circular dichroism – A (or ) = AL – AR ~ Im (•m) = R01

Trick – measure difference since A is small ~10-3

-10-4

:

5

Circular Dichroism

Theoretically this ~ R = Im [( ex m g) ( ex g)]

electronic dipole operator

m magnetic dipole operator

m 0 only for chiral molecules e.g. asymmetric C / not superimposable on mirror image/ no plane or center of symmetry opposite enantiomer, flip sign

“Perfect for biology” “all” bio-molecular, chiral

i.e. proteins L – AA – amide * and aromatics

DNA chiral ribose, bases great in UV, all in IR

sugars several centers –lack chromophore

lipids well …can be chiral, few chromophores

6

Structures: chiral polymers (helicity or twist), select - local L,D

Measurement of CD is most widely used for protein secondary structure

-helix - most intense, 222 & 207nm

-sheet - weaker, neg 215 nm, pos 200 nm coil – weak or pos near UV, neg <200 nm

7

DNA – band patterns vary – more transitions, base depend Big success: B vs. Z differ: right – left handed helices

Examples are G-C oligos, A-T a little different

Sugars – problem, absorbance bands in vacuum UV Lipids -- similar issues, also conformations less restricted

ORD - like measuring index of refraction no absorption Can measure optical rotation in “clear” sample (sugar)

= ()(nL – nR) where nL & nR index in circ. light circular birefringence

CD is absorption spectra, so need absorption band

A = AL –AR but can be measured/expressed as ellipticity

(degrees) = 32.99 A

alternate:- molar ellipticity scale ( l – path(cm or dm)):

[] = Cl = 3300 -- normal for biochem spectra

Can convert CD ↔ ORD – Transform: integrate over all

8

In IR can do VCD, called Vibrational Circular Dichroism Signals smaller (need more concentration) but

differentiation between states/conformations is higher

VCD measures same transitions as IR, but has shape/sign

Patterns for VCD discriminate helices, sheets and coils Also distinguish 310-helices, turns and other structures Coil shown to be characterized by left hand turns

due to similarity with poly L-Pro II helices

9

Derivative shape from coupling of dipoles (CD and VCD)

DNA VCD - base region sensitive to G-C to A-T ratio,

–PO2 region independent, RNA ~same, ex. sym –PO2 Easily sense B- and Z-form DNA, A- similar to B-form

10

Triplex DNA has unique pattern

Also can use isotopes to localize structural information