Continued from part a

1
Continued from part a
2
Also Raman
Not Raman, unless RR
Weak IR Multiple bands
3
Peptide conformation depends on f, y angles
If (f,y) repeat, they determine secondary
structure
Chromophores amides are locally achiral CD has
little signal without coupling, ideal for
detection -- IR, Raman resolve shift
Detection requires method sensitive to amide
coupling
Far UV absorbance broad, little
fluorescencecoupling impact small
4
Model polypeptide IR absorbance spectra - Amide I
and II
(Not in Raman)
(weak IR but strong 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 lt 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 AAn oligomers VCD have DA/A const
with n vibrational (Force Field) coupling plus
dipole coupling Development -- structure-spectra
relationships Small molecules theory /
Biomolecules -- empirical, Recentpeptide 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 Radiation and Right Incident
and/or Scattered Radiation
7
 
UIC Dispersive VCD Schematic
Yes it still exists and measures VCD!
Electronics
Optics and Sampling
8
Separate VCD Bench
Optics FTIR
UIC FT-VCD Schematic (designed for magnetic VCD
commercial ones simpler)
Electronics
Polarizer PEM (ZnSe) Sample
detector
FT-computer
Optional magnet
Detector (MCT)
filter
lock-in amp
PEM ref
9
Large electric dipole transitions can couple over
longer ranges to sense extended conformation
Simplest representation is coupled oscillator
De eL-eR
Dipole coupling results in a derivative shaped
circular dichroism
l
Real systems - more complex interactions - but
pattern is often consistent
10
Selected model Peptide VCD, aqueous solution
11
Nature of the peptide random coil form
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
Dukor and Keiderling 1991 with ECD, VCD, and IR
showed Pron 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
Reference Poly(Lys) coil, pH 7
ECD of Pron oligomers
Dukor, Keiderling - Biopoly 1991
Greenfield Fasman 1969
13
Relationship to random coil - compare Pron and
Glun
IR same, VCD - same shape, half size --
partially ordered
Dukor, Keiderling - Biopoly 1991
14
Thermally unfolding random coil poly-L-Glu -IR,
VCD
VCD loses magnitude
T 5oC (___) 25oC (- - -) 75oC (-.-.-)
IR shifts frequency
random coil must have local order
Keiderling. . . Dukor, Bioorg-MedChem 1999
15
Comparison of Protein VCD and IR
VCD in H2O
FTIR in H2O
a
b
a/b
Wavenumbers (cm-1)
16
VCD Example ?- vs. the 310-Helix
?-Helix
310-Helix
i, i4 ? H-bonding ? i, i3 3.6 ?
Res./Turn ? 3.0 2.00 ? Trans./Res (Å)
? 1.50
17
The VCD success example 310-helix vs. a-helix
i-gti3
Aib2LeuAib5
310
mixed
a
(Met2Leu)6
i-gti4
Relative shapes of multiple bands distinguish
these similar helices
Silva et al. Biopolymers 2002
18
Simulated IR and VCD spectra The best practical
computations for the largest possible molecules
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
19
Transfer of FF, APT and AAT (e.g. Ala7 to Ala20)
Method from Bour et al. J. Comp Chem. 1997
Main chain residues
20-mer
Middle residue
C-terminus
N-terminus
7-mer FF, APT, AAT calculated at BPW91/6-31G
level
Kubelka, Bour, et al., ACS Symp. Ser.810, 2002
20
Uniform long helices?characteristic, narrow bands
Simulations
7-amide disperse amide I, II bands
vacuum
D2O
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
Frequency error mostly solvent origin
21
Simulation of Helix IR and VCD Really Works!
Experiment
Simulation 310-helix
310-helix vs. a-helix comparison of Aibn, Alan
and (Aib-Ala)n sequences.
Aib
2
5
Simulation a-helix
4
in CDCl
(Met
-Leu)
2
8
1500
1600
1700
-1
Wavenumber cm

(Kubelka,Silva, Keiderling JACS 2002)
22
Isotopic Labeling old technique - new
twist Shift frequency by n (k/m)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
a-helix model Alanine 20-mer 13C labeling scheme
Silva, Kubleka, et al. PNAS 2000
24
a-helix
ProII-like
Simul.
High T
Low T
Exper.
Simulated and experimental IR absorption for
Ala20 with 13C labels C-term is different, do not
know structure from IR
Silva, Kubleka, et al. PNAS 2000
25
ProII-like
a-helix
High T
Low T
Simulated and experimental VCD for Ala20 with 13C
labels VCD shows helical at all but C-terminal,
where it is coil
Silva, Kubleka, et al. PNAS 2000
26
a
b
c
d
Wavenumber cm-1
Temperature dependent Ala20 VCD a)
unlabeled b) C-terminus c) N-terminus d)
Middle(N) labeled
27
Unstable termini VCD identify location - isotope
Frequency shift of 12C 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
28
Monomeric b-sheet models hairpins 13CO
labeling - sense cross-strand coupling
Setnicka et al. JACS 2005
29
Two labeling types, distinct cross-strand coupling
Simulation
Experiment
Setnicka et al. JACS 2005
30
IR spectra of labeled Gellman A peptide heating
from 5 (violet) to 85C (red), step 5C
Hairpin labeling works - Site-specific folding
Major unfolding impact on 13CO, loss of coupling
IR
Setnicka, et al. unpublished
31
VCD of DNA, vary A-T to G-C ratio
DNA
base deformations
sym PO2- stretches
-1
big variation
little effect
32
DNA VCD of PO2- modes in B- to Z-form transition
B, A
B
Z
Z
A
B
DNA
Experimental
Theoretical
33
Triplex DNA, RNA form by adding third strand to
major groove with Hoogsteen base pairing
DNA
34
VCD of Triplex formationbase modes
CGC
-20
DNA
Wavenumber (cm-1)
35

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