1. Continued from part a

2. Also Raman Not Raman, unless RR Weak IR Multiple bands

3. Peptide conformation depends on ,  angles Far UV absorbance broad, little fluorescence—coupling impact small Detection requires method sensitive to amide coupling If (  repeat, they determine secondary structure Chromophores – amides are locally achiral CD has little signal without coupling, ideal for detection -- IR, Raman resolve shift

4. I II Model polypeptide IR absorbance spectra - Amide I and II (weak IR but strong in Raman) (Not in Raman)

5. Combining Techniques: Vibrational CD “CD” in the infrared region Probe chirality of vibrations  goal stereochemistry Many transitions / Spectrally resolved / Local probes Technology in place -- separate talk Weak phenomenon - limits S/N / Difficult < 700 cm -1 Same transitions as IR same frequencies, same resolution Band Shape from spatial relationships neighboring amides in peptides/proteins Relatively short length dependence AA n oligomers VCD have  A/A ~ const with n vibrational (Force Field) coupling plus dipole coupling Development -- structure-spectra relationships Small molecules – theory / Biomolecules -- empirical, Recent—peptide VCD can be simulated theoretically

6. VIBRATIONAL OPTICAL ACTIVITY Differential Interaction of a Chiral Molecule with Left and Right Circularly Polarized Radiation During Vibrational Excitation VIBRATIONAL CIRCULAR DICHROISM RAMAN OPTICAL ACTIVITY Differential Absorption of Left and Right Differential Raman Scattering of Left Circularly Polarized Infrared Radiationand Right Incident and/or Scattered Radiation

7. UIC Dispersive VCD Schematic Electronics Optics and Sampling Yes it still exists and measures VCD!

8. UIC FT-VCD Schematic (designed for magnetic VCD commercial ones simpler) Electronics Optics FTIR Separate VCD Bench Polarizer PEM (ZnSe) Sample Detector (MCT) Optional magnet lock-in amp filter PEM ref detector FT-computer

9. Large electric dipole transitions can couple over longer ranges to sense extended conformation Simplest representation is coupled oscillator T ab aa bb Real systems - more complex interactions - but pattern is often consistent Dipole coupling results in a derivative shaped circular dichroism   L  R

10. Selected model Peptide VCD, aqueous solution Amide I Amide II   coil AA

11. Tiffany and Krimm in 1968 noted similarity of Proline II and poly-lysine ECD and suggested “extended coil” Problem -- CD has local sensitivity to chiral site --IR not very discriminating Nature of the peptide random coil form Dukor and Keiderling 1991 with ECD, VCD, and IR showed Pro n oligomers have characteristic random coil spectra Suggests -- local order, left-handed turn character -- no long range order in random coil form Same spectral shape found in denatured proteins, short oligopeptides, and transient forms

12. Dukor, Keiderling - Biopoly 1991 ECD of Pro n oligomers Reference: Poly(Lys) – “coil”, pH 7 Greenfield & Fasman 1969 Builds up to Poly-Pro II frequency --> tertiary amide helix sheet ‘coil’ Single amide

13. Dukor, Keiderling - Biopoly 1991 Relationship to “random coil” - compare Pro n and Glu n IR ~ same, VCD - same shape, half size -- partially ordered

14. Thermally unfolding “random coil” poly-L-Glu -IR, VCD T = 5 o C ( ___ ) 25 o C (- - -) 75 o C (-.-.-) VCD loses magnitude IR shifts frequency “random coil” must have local order Keiderling... Dukor, Bioorg-MedChem 1999

15. VCD in H 2 OFTIR in H 2 O Wavenumbers (cm -1 ) Comparison of Protein VCD and IR   

16. VCD Example:  - vs. the 3 10 -Helix i, i+4  H-bonding  i, i+3 3.6  Res./Turn  3.0 2.00  Trans./Res (Å)  1.50  -Helix 3 10 -Helix

17. The VCD success example: 3 10 -helix vs.  -helix Relative shapes of multiple bands distinguish these similar helices Aib 2 LeuAib 5 (Met 2 Leu) 6  3 10 mixed i  i+3 i  i+4 Silva et al. Biopolymers 2002

18. 1. Ab Initio (DFT) quantum mechanical calculations can give necessary data for small molecules Frequencies from force field -diagonalize second derivatives of the energy Intensities from change in dipole moment with motion Express all as atomic properties 2. Large bio-macromolecules --need a trick (Bour et al. JCompChem 1997) Transfer atomic properties from “small” model In our case these “small” calculations are some of the largest peptides ever done ab initio Simulated IR and VCD spectra The best practical computations for the largest possible molecules

19. Transfer of FF, APT and AAT (e.g. Ala 7 to Ala 20 ) Main chain residues Middle residue N-terminus C-terminus 20-mer 7-mer: FF, APT, AAT calculated at BPW91/6-31G* level Kubelka, Bour, et al., ACS Symp. Ser.810, 2002 Method from Bour et al. J. Comp Chem. 1997

20. Uniform long helices  characteristic, narrow bands vacuum D2OD2O 7-amide disperse amide I, II bands 21-amide: narrow IR band by change intensity distribution, preserve mode dispersion and VCD shape, solvent -- close amide I-II gap Kubelka & Keiderling, J.Phys.Chem.B 2005 Simulations Frequency error mostly solvent origin

21. in CDCl in TFE (Aib-Ala) 4 Wavenumber [cm ] 150016001700 Aib 5 -Leu-Aib 2 (Met 2 -Leu) 8 3 10 -helix vs.  -helix: comparison of Aib n, Ala n and (Aib-Ala) n sequences. Simulation:  -helix Experiment:Simulation: 3 10 -helix Simulation of Helix IR and VCD Really Works! (Kubelka,Silva, Keiderling JACS 2002)

22. Isotopic Labeling – old technique - new twist Shift frequency by ~ (k/  ) 1/2 Tends to decouple from other modes, and can disrupt their exciton coupling Not intense, compare to polymer repeat Isolated oscillator (transition) in other modes Requirement: High S/N, good baseline focus on one band  dispersive VCD?

23.  -helix model: Alanine 20-mer 13 C labeling scheme Silva, Kubleka, et al. PNAS 2000

24. Simulated and experimental IR absorption for Ala 20 with 13 C labels C-term is different, do not know structure from IR  -helix ProII-like Low T High T Silva, Kubleka, et al. PNAS 2000 Simul. Exper.

25.  -helix ProII-like Low T High T Simulated and experimental VCD for Ala 20 with 13 C labels VCD shows helical at all but C-terminal, where it is “coil” Silva, Kubleka, et al. PNAS 2000

26. Wavenumber [cm -1 ] ab cd Temperature dependent Ala 20 VCD: a) unlabeled b) C-terminus c) N-terminus d) Middle(N) labeled

27. Frequency shift of 12 C amide I’ VCD band minimum with temperature: a) terminal, b) middle labeled. Unlabeled added for comparison. Termini “melt” at lower temperatures Silva, Kubleka, et al. PNAS 2000 Unstable termini – VCD identify location - isotope

28. Monomeric  -sheet models – hairpins 13 C=O labeling - sense cross-strand coupling Setnicka et al. JACS 2005

29. Two labeling types, distinct cross-strand coupling SimulationExperiment Setnicka et al. JACS 2005

30. IR spectra of labeled Gellman A peptide: heating from 5 (violet) to 85  C (red), step 5  C labeled on Val3 and Lys8 Lys Hairpin labeling works - Site-specific folding IR Setnicka, et al. unpublished Major unfolding impact on 13 C=O, loss of coupling

31. VCD of DNA, vary A-T to G-C ratio base deformationssym PO 2 - stretches big variation little effect

32. A B DNA VCD of PO 2 - modes in B- to Z-form transition ExperimentalTheoretical Z B B, A Z

33. Triplex DNA, RNA form by adding third strand to major groove with Hoogsteen base pairing

34. -20 CGC+ Wavenumber (cm -1 ) VCD of Triplex formation—base modes

35. That is all for now Good luck on exams I enjoyed having you in class this Fall Tim Keiderling