continued from part a
DESCRIPTION
Continued from part a. Also Raman. Not Raman, unless RR. Weak IR Multiple bands. 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 - PowerPoint PPT PresentationTRANSCRIPT
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Continued from part a
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Characteristic Amide Vibrations
I - Most useful;IR intense, less interference (by solvent, other modes,etc)
Less mix (with other modes)
II - IR intense
III - Raman Intense
A – often obscuredby solvent
IV – VII – difficult to detect, discriminate
~3300 cm-1
~1650 cm-1
1500-50 cm-1
1300-1250 cm-1
700 cm-1
mix
Also Raman
Not Raman, unless RR
Weak IR Multiple bands
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Peptide conformation depends on , angles
Far UV absorbance broad, little fluorescence—coupling impact smallDetection 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
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I
II
Model polypeptide IR absorbance spectra - Amide I and II
(weak IR but strong in Raman)
(Not in Raman)
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Combining Techniques: Vibrational CD “CD” in the infrared region
Probe chirality of vibrations goal stereochemistryMany transitions / Spectrally resolved / Local probes
Technology in place -- separate talkWeak phenomenon - limits S/N / Difficult < 700 cm-1
Same transitions as IR same frequencies, same resolution
Band Shape from spatial relationshipsneighboring amides in peptides/proteins
Relatively short length dependenceAAn oligomers VCD have A/A ~ const with n
vibrational (Force Field) coupling plus dipole couplingDevelopment -- structure-spectra relationships
Small molecules – theory / Biomolecules -- empirical, Recent—peptide VCD can be simulated theoretically
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VIBRATIONAL OPTICAL ACTIVITYVIBRATIONAL OPTICAL ACTIVITYDifferential 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
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G
C F
M2S
M1
PEMP
SCL
D
D Pre-Amp
DynamicNormalization
TunedFilter
Lock-in
C
Lock-in
Chopper ref. C
PEM ref.
M
TransmissionFeedback Lock-in
A/DInterface
ComputerInterfaceMonochromator
UIC Dispersive VCD Schematic
Electronics
Optics and Sampling
Yes it still exists and measures VCD!
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UIC FT-VCD
Schematic(designed for magnetic VCD commercial ones simpler)
Electronics
Optics FTIR
Separate VCD Bench
PolarizerPEM (ZnSe)Sample
Detector (MCT)
Optional magnet
lock-in ampfilter PEM ref
detector
FT-computer
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Large electric dipole transitions can couple over longer ranges to sense extended conformation
Simplest representation is coupled oscillator
Taba
b
Real systems - more complex interactions- but pattern is often consistent
baabTc
2
πR
Dipole coupling results in a derivative shaped circular dichroism
LR
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Selected model Peptide VCD, aqueous solution
Amide IAmide II
coil
A
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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 Pron oligomers have characteristic random coil spectraSuggests -- 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
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Dukor, Keiderling - Biopoly 1991
ECD of Pron oligomers Reference: Poly(Lys) – “coil”, pH 7
Greenfield & Fasman 1969
Builds up to Poly-Pro II frequency --> tertiary amide helix
sheet
‘coil’
Single amide
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Dukor, Keiderling - Biopoly 1991
Relationship to “random coil” - compare Pron and Glun
IR ~ same, VCD - same shape, half size -- partially ordered
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Thermally unfolding “random coil” poly-L-Glu -IR, VCD
T = 5oC (___) 25oC (- - -) 75oC (-.-.-)
VCD loses magnitude
IR shifts frequency
“random coil” must have local order
Keiderling. . . Dukor, Bioorg-MedChem 1999
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VCD in H2OFTIR in H2O
Wavenumbers (cm-1)
Comparison of Protein VCD and IR
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VCD Example: - vs. the 310-Helix
i, i+4 H-bonding i, i+3
3.6 Res./Turn 3.0
2.00 Trans./Res (Å) 1.50
-Helix 310-Helix
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The VCD success example: 3The VCD success example: 31010-helix vs. -helix vs. -helix-helix
Relative shapes of multiple bands distinguish these similar helices
Aib2LeuAib5
(Met2Leu)6
310
mixed
ii+3
ii+4
Silva et al. Biopolymers 2002
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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 motionExpress all as atomic properties
2. Large bio-macromolecules--need a trick (Bour et al. JCompChem 1997)
Transfer atomic properties from “small” modelIn our case these “small” calculations are some of the
largest peptides ever done ab initio
Simulated IR and VCD spectraThe best practical computations for the largest possible molecules
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Transfer of FF, APT and AAT (e.g. Ala7 to Ala20)
Main chain residues
Middle residueN-terminus C-terminus
20-mer
7-mer: FF, APT, AAT calculated at BPW91/6-31G* levelKubelka, Bour, et al., ACS Symp. Ser.810, 2002
Method from Bour et al. J. Comp Chem. 1997
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Uniform long helicescharacteristic, narrow bands
vacuum D2O
7-amide disperse amide I, II bands
21-amide: narrowIR 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
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in CDCl
in TFE(Aib-Ala)4
Wavenumber [cm-1
]
150016001700
Aib5-Leu-Aib 2
(Met2-Leu) 8
310-helix vs. -helix:
comparison of Aibn,
Alan and (Aib-Ala)n sequences. Simulation: -helix
Experiment: Simulation: 310-helixSimulation of Helix IR and VCD Really Works!
(Kubelka,Silva, Keiderling JACS 2002)
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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?
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-helix model: Alanine 20-mer 13C labeling scheme
Notation Label position Peptide sequence
unlabeled none Ac-AAAAKAAAAKAAAAKAAAAY-NH2
L1 N-terminus Ac-AAAAKAAAAKAAAAKAAAAY-NH2
L2 Middle (closer to N-terminus) Ac-AAAAKAAAAKAAAAKAAAAY-NH2
L3 Middle (closer to C-terminus) Ac-AAAAKAAAAKAAAAKAAAAY-NH2
L4 C-terminus Ac-AAAAKAAAAKAAAAKAAAAY-NH2
Silva, Kubleka, et al. PNAS 2000
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Simulated and experimental IR absorption for Ala20 with 13C labelsC-term is different, do not know structure from IR
-helix ProII-like
Low T High T
Silva, Kubleka, et al. PNAS 2000
Simul.
Exper.
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-helix ProII-like
Low T High T
Simulated and experimental VCD for Ala20 with 13C labelsVCD shows helical at all but C-terminal, where it is “coil”
Silva, Kubleka, et al. PNAS 2000
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Wavenumber [cm-1]
a b
c d
Temperature dependent Ala20 VCD: a) unlabeled b) C-terminus c) N-terminus d) Middle(N) labeled
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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
Unstable termini – VCD identify location - isotope
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small H- bonding ring large H-bonding ring
Monomeric -sheet models – hairpins13C=O labeling - sense cross-strand coupling
Setnicka et al. JACS 2005
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Two labeling types, distinct cross-strand coupling
Simulation Experiment
Setnicka et al. JACS 2005
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IR spectra of labeled Gellman A peptide: heating from 5 (violet) to 85C (red), step 5C
Wavenumber, cm-1
160016501700
0.0
0.2
0.4
0.6
A
labeled on Val3 and Lys8
NH
NH
NH
NH
NH
NH3
+
Arg
O TyrNH
NH
NH
NH
NH
NH
Gln O O
OO
O
O
O
O
OO
Val
Glu
Val
Leu
Ile
Lys
Orn
NH2
O
Lys
Hairpin labeling works - Site-specific folding
IR
Setnicka, et al. unpublished
Major unfolding impact on 13C=O, loss of coupling
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-1
VCD of DNA, vary A-T to G-C ratiobase deformations sym PO2
- stretches
big variation little effect
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A B
DNA VCD of PO2- modes in B- to Z-form transition
Experimental Theoretical
Z
B B, A
Z
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Triplex DNA, RNA form by adding third strand to major groove with Hoogsteen base pairing
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-20CGC+
Wavenumber (cm-1)
VCD of Triplex formation—base modes
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• That is all for now
• Good luck on exams
• I enjoyed having you in class this Fall
• Tim Keiderling