Polarization and Circular Dichroism - 2014 (Notes 17) Since is … 17 (Circular... ·...
Transcript of Polarization and Circular Dichroism - 2014 (Notes 17) Since is … 17 (Circular... ·...
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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)
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–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
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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,
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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
:
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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
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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
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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
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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
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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
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Triplex DNA has unique pattern
Also can use isotopes to localize structural information