Polarization and Circular Dichroism (Notes 17)
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1 Polarization and Circular Dichroism (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 p x in H-atom, excite by E x x, (but not orient!) * in ethylene, C 2 H 4, 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) 1
2 Alternate: alkyl tail, hydrophilic res. bind surface charges (PO 2 - ) - amphipathic helices (+/- sides) can lay on surface, hydrophobic penetrate, charges out or form assemblieshelical 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 2
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 In plane, Out of plane, 3
4 Circular Polarization Phase shift - 2 E-M waves displaced by /4 along z combine rotates E as propagate (trace helix, L - R) Make by mix two beams, shift phase in birefringent crystal Circular Polarization Right or Left use birefringent plate with n x n y - retard (slow) E x or E y one polarization relative to other, tune: adjust path or stress Molecule sees both linear polarizations (E x + E y ) oscillate but due to the rotation between them at (circularity) has different selection rules Chiral molec, A L A R Circular dichroism A (or ) = A L A R ~ Im ( m) = R 01 Trick measure difference since A is small ~ : 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 5
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 6
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) = ( )(n L n R ) where n L & n R index in circ. light circular birefringence CD is absorption spectra, so need absorption band A = A L A R but can be measured/expressed as ellipticity (degrees) = A alternate: - molar ellipticity scale ( l path(cm or dm)): [ ] = Cl = normal for biochem spectra Can convert CD ORD Transform: integrate over all 7
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 helices, turns and other structures Coil shown to be characterized by left hand turns due to similarity with poly L-Pro II helices 8
9 Derivative shape from coupling of dipoles (CD and VCD) DNA VCD - base region sensitive to G-C to A-T ratio, PO 2 region independent, RNA ~same, ex. sym PO 2 Easily sense B- and Z-form DNA, A- similar to B-form 9
10 Triplex DNA has unique pattern Also can use isotopes to localize structural information 10
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