ΔA(λ) = A(λ)LCPL - A(λ)RCPL Circular Dichroism ΔA(λ) = A(λ)LCPL - A(λ)RCPL

Polarized Light Light with oscillations confined to a single plane. Top: vertically polarized light Bottom: horizontally polarized light Videos throughout notes from http://www.photophysics.com/polarization.php

Circularly Polarized Light Sum of vertically and horizontally plane-polarized light in which the phases differ by a quarter wave Does this video represent left or right-handed circularly polarized light? Draw separate 2D representations for the vertical and horizontal components that clearly show the phase difference. Diagram the phase different required to obtain the opposite direction of circularly polarized light.

Circular Dichroism (CD) Spectroscopy Difference in absorption between left-handed and right-handed circularly polarized light as a function of wavelength Unlike optical rotatory dispersion (ORD) spectroscopy, which only requires a chiral molecule, CD requires a chromophore in a chiral environment.

ORD – why isn’t a chromophore required? All chiral molecules exhibit circular birefringence, basically the rates of propagation of left and right-handed polarized light (which can be added to form plane-polarized light) are different. This changes their relative phase, and therefore the observed angle at the detector. With the background given so far, how would the above video change to represent CD?

Origin of Ellipticity Unit Would the signal above be positive or negative?

Chromophores in Biomolecules Proteins Aromatic sidechains (π-π* ~280 nm, near UV) Phenylalanine (ε ~ 250 M-1cm-1) Tyrosine (ε ~ 1000 M-1cm-1) Tryptophan (ε ~ 5000 M-1cm-1) Backbone amide bond (far UV) n-π* ~210-220 nm (major peak in UV spectrum, (ε ~ 100 M-1cm-1) π-π* ~190 nm Sensitive to tertiary structure Sensitive to secondary structure

UV Absorbance vs. CD: Poly-L-lysine Random coil: positive CD @ 216nm negative CD @ 198nm Beta-sheet: negative CD @ 218nm positive CD @ 195nm Alpha-helix: negative CD @ 222nm negative CD @ 208nm positive CD @ 192nm 3 peaks from 2 electronic transitions? π-π* n-π* UV Absorbance: Cantor & Schimmel, 1980 CD: Campbell & Dwek, 1984 Both images from http://www.cryst.bbk.ac.uk/PPS2/course/section8/ss-960531_21.html

Delocalized Excitation (Exciton) Organic conformational analysis and stereochemistry from circular dichroism spectroscopy, Lightner and Gurst, Wiley, 2000.

Spectroscopic Impact of Excitonic Coupling

Excitonic Coupling of Multiple Chromophores with Similar Excitation Energies (Examples) head-to-tail dipole orientation sandwich dipole orientation 800 nm 780 nm 820 nm 800 nm 790 nm 810 nm From http://www.chem.vu.nl/~zwan/lectures/Egmond_3.ppt

Why is Excitonic Coupling Dependent on Secondary Structure?

CD: Reflection of Environment on Protein Folding Which traces suggest random coil structure? What secondary structure type is suggested by the other traces? Can you estimate the fraction random coil or other secondary structure?

Near-UV CD Data Analysis Deconvolution: determining contributions to observed spectrum from separate secondary structures Algorithms (most available through Dichroweb) Singular value decomposition: determines coefficients for a matrix of basis spectra of known secondary structure Self-consistent (SELCON3): initial guess by SVD followed by iteration to self-consistent result K2D: neural network trained using CD/structural data on reference proteins to translate input CD spectra to secondary structure proportions Dichroweb: http://dichroweb.cryst.bbk.ac.uk/

Peptide Design Goal: ~60% helix Tris-HCl (mM) Ethanol (%) TFE (%) °C Helix (%) Strand (%) Turns (%) Unordered (%) Total (%) 5 7 9 77 100 50 22 15 14 101 10 36 12 18 34 20 44 16 30 3 99 How well did you estimate?

Other Uses of CD – Thermal Stability Unfolded fraction (monitored at 222nm) = (θobs - θdenatured)/(θfolded - θdenatured) Isoform Melting Point (Tm), °C Untreated EDTA Ca2+ Co2+ Ni2+ Zn2+ NPP2 58, 86 56, 102 56, 86 56,88 80 58 NPP6 76 70 N/A 68

Other Uses of CD – Absolute Stereochemistry of Chromophores Organic conformational analysis and stereochemistry from circular dichroism spectroscopy, Lightner and Gurst, Wiley, 2000.

Data Interpretation Problem Given: p-Dimethylaminobenzoate has an electronic transition dipole running through the substituents with a UV εmax,310 of ~30,000. One stereoisomer of the bis-p-dimethylaminobenzoate derivative of trans-1,2-cyclohexanediol shows CD Δεmax,295 of -44 and Δεmax,320 of +83. Which stereoisomer is this? (give both +/- chirality and R/S designation of both stereocenters)

Solution