Download Continued from part a

Continued from part a
Characteristic Amide Vibrations
~3300 cm-1
A – often obscured
by solvent
I - Most useful;
~1650 cm-1 IR intense, less interference
(by solvent, other modes,etc)
Less mix (with other modes)
Also Raman
1500-50 cm-1 II - IR intense
mix
Not Raman, unless RR
III - Raman Intense
1300-1250 cm-1 Weak IR Multiple bands
700 cm-1
IV – VII – difficult
to detect, discriminate
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 fluorescence—coupling impact small
Model polypeptide IR absorbance spectra bsorbance spectra of selected model peptides
Amide I and II
I
Absorbance
3
helix
II
2
-structure
(Not in Raman)
1
random
coil
0
1750 1700 1650 1600 1550 1500 1450
-1
Wavenumbers (cm )
(weak IR but
strong in Raman)
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
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,
Recent—peptide VCD can be simulated theoretically
VIBRATIONAL OPTICAL ACTIVITY
Differential Interaction of a Chiral Molecule with Left and Right Circularly
Polarized Radiation During Vibrational Excitation
VIBRATIONAL CIRCULAR DICHROISM
Differential Absorption of Left and Right
Circularly Polarized Infrared Radiation
RAMAN OPTICAL ACTIVITY
Differential Raman Scattering of Left
and Right Incident and/or Scattered
Radiation
UIC Dispersive VCD Schematic
Yes it still exists and measures VCD!
Electronics
D
PreAmp
Dynamic
Normalization
C
Lock-in

Lock-in
Tuned
Filter
Transmission
Feedback Lock-in
PEM ref.
M
Chopper ref. C
G
C
S
Monochromator
Interface
A/D
Interface
Computer
Optics and Sampling
F
M2
M1
L
P
SC
PEM
D
Optics Separate VCD Bench
UIC
FTIR
FT-VCD
Schematic
(designed for
magnetic VCD
commercial
ones simpler)
Electronics
Polarizer
PEM (ZnSe)
Sample
detector
FT-computer
filter
lock-in amp
PEM ref
Optional magnet
Detector (MCT)
Large electric dipole transitions can couple over
longer ranges to sense extended conformation
Simplest representation is coupled oscillator
 π
R  
 2c




Tab  m a  m b )

De 
eL-eR
l
ma
Tab
mb
Dipole coupling
results in a
derivative shaped
circular dichroism
Real systems - more complex interactions
- but pattern is often consistent
Selected
VCD spectra
Selected
model model
Peptidepeptide
VCD, aqueous
solution
Amide I
30
DA
helix
Amide II
a
VCD (A. U.)
20

10
-structure
0
random
coil
-10
coil
1750 1700 1650 1600 1550 1500 1450
Wavenumbers (cm -1)
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
ECD of Pron oligomers
Reference: Poly(Lys)
– “coil”, pH 7
Single
amide
Builds up to
Poly-Pro II
frequency -->
tertiary amide
Dukor, Keiderling - Biopoly 1991
sheet
‘coil’
helix
Greenfield & Fasman 1969
Relationship to “random coil” - compare Pron and Glun
IR ~ same, VCD - same shape, half size -- partially ordered
Dukor, Keiderling - Biopoly 1991
Thermally unfolding “random coil” poly-L-Glu -IR, VCD
T = 5oC (___)
25oC (- - -)
75oC (-.-.-)
“random coil”
must have
local order
VCD loses
magnitude
IR shifts
frequency
Keiderling. . . Dukor, Bioorg-MedChem 1999
IR absorbance
spectra of
some
Comparison
of Protein
VCD
and IR
selected proteins in H2O
Vibrational Circular Dichroism spectra o
VCD in H2O in H O
some selected proteins
FTIR-Deconvolved
2
FTIR
in H2O
FTIR
HEM
a
HEM
HEM
CON
A
LYS

A
DA CON
LYS
a/
1700 1650 1600 1550 1500
Wavenumbers (cm-1)
CON
LYS
17001700
16501650
16001600
15501550
15001500
-1
Wavenumbers (cm )
VCD Example: a- vs. the 310-Helix
a-Helix
i, i+4  H-bonding  i, i+3
3.6 
Res./Turn
 3.0
2.00  Trans./Res (Å)  1.50
310-Helix
The VCD success example: 310-helix vs. a-helix
4
310-helical
i->i+3
500
Aib2LeuAib5
310-helical
400
310
2
(Aib-Ala)6
1
a-helical
(Met2Leu)6
0
1800
Ala(AibAla)3
300
Ala(AibAla)3
DA (A.U.)
Absorbance
3
1600
-1
Wavenumbers (cm )
1400
200
(Aib-Ala)6
100
mixed
0
a
-100
i->i+4
1800
1600
a-helical
1400
Wavenumbers (cm-1)
Relative shapes of multiple bands distinguish these similar helices
Silva et al. Biopolymers 2002
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
Transfer of FF, APT and AAT (e.g. Ala7 to Ala20)
Method from Bour et al. J. Comp Chem. 1997
20-mer
N-terminus
Main chain residues
Middle
residue
C-terminus
7-mer: FF, APT, AAT calculated at BPW91/6-31G* level
Kubelka, Bour, et al., ACS Symp. Ser.810, 2002
Uniform long helicescharacteristic, narrow bands
d
a
Simulations
2
0
-2
2
De' (x10e'
) (x10-2)
De' (x102)
-4
2
3
0
22
-2
10
-4
-2
2
b
a
a
De' (x102)
d
D 2O
c
f
-4
3
b
e
0
2
a
d
22
b
e
-2
0
10
-2
-4
-2
-4 1800 1700 1600 1500 1400 1300 1200 1800 1700 1600 1500 1400 1300 1200
-4
3
c
e' (x10-2)
vacuum
ed
f
2
Wavenumber
(cm-1)
3
b
e
a
22
c
fd
0
20
1
-2
-2
1
-4
-4 1800 1700 1600 1500 1400 1300 1200 1800 1700 1600 1500 1400 1300 1200
2
c
3
Wavenumberf (cm-1)
b 1700 1600 1500 1400 1300
1800
1200 1800
e 1700 1600 1500 1400 1300 1200
0
2
Wavenumber (cm-1)
-2
Frequency error mostly solvent origin
1
-4
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
Simulation of Helix IR and VCD Really Works!
310-helix vs. a-helix:
comparison of Aibn,
Alan and (Aib-Ala)n
sequences.
Experiment:
Aib5-Leu-Aib
Simulation: 310-helix
Ac-(Aib)8-NH2
2
Simulation: a-helix
(Aib-Ala) 4
Ac-(Aib-Ala)3-NH2
Ac-(Aib-Ala)4-NH2
in TFE
in CDCl
De/amide
De/amide
1700
Ac-(Ala)8-NH2
(Met 2-Leu) 8
Ac-(Ala)6-NH2
1600
1500
-1
Wavenumber [cm ]
1700
1600
Wavenumber [cm
1500
-1
]
1700
1600
1500
Wavenumber [cm-1]
(Kubelka,Silva, Keiderling JACS 2002)
Isotopic Labeling – old technique - new twist
Shift frequency by  ~ (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?
a-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
a-helix
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
e x 10-3)
4
ProII-like
Simul.
2
1750
12
Anorm (x 10)
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
1700
1650
1750
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
Low T
1700
1650
Unalbeled
N-terminus
C-terminus
Middle (N)
Middle (C)
High T
Exper.
8
4
0
1700
1650
1600
Wavenumber [cm-1]
1550
1700
1650
1600
1550
Wavenumber [cm-1]
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
2
De x 10)
0
a-helix
ProII-like
-2
-4
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
-6
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
-8
1750
1700
1650
4
5
1700
1650
High T
Low T
DAnorm (x 10 )
1750
0
-4
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
-8
1700
1650
1600
Wavenumber [cm-1]
1550
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
1700
1650
1600
1550
Wavenumber [cm-1]
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
4
a
b
DA (x105)
0
5 deg
10 deg
15 deg
20 deg
25 deg
30 deg
35 deg
40 deg
45 deg
50 deg
55 deg
60 deg
-4
-8
4
c
d
DA (x105)
0
-4
-8
1660
1620
1580
1660
1620
1580
Wavenumber [cm-1]
Temperature dependent Ala20 VCD:
a) unlabeled b) C-terminus c) N-terminus d) Middle(N) labeled
Unstable termini – VCD identify location - isotope
a
1653
b
Unlabeled
Middle (C)
Middle (N)
C-terminus
N-terminus
Unlabeled
Frequency [cm-1]
1651
1649
1647
1645
1643
10
20
30
40
50
60
Temperature [oC]
12C amide I’ VCD
10
20
30
40
50
60
Temperature [oC]
Frequency shift of
band minimum with temperature:
a) terminal, b) middle labeled. Unlabeled added for comparison.
Termini “melt” at lower temperatures Silva, Kubleka, et al. PNAS 2000
Monomeric -sheet models – hairpins
13C=O labeling - sense cross-strand coupling
small H- bonding ring
large H-bonding ring
Setnicka et al. JACS 2005
Two labeling types, distinct cross-strand coupling
Simulation
Experiment
Setnicka et al. JACS 2005
Hairpin labeling works - Site-specific folding
0.6
Major unfolding
impact on 13C=O,
loss of coupling
IR
A
0.4
Arg
+
0.2
H3N
H
N
O
O
H2N
1700
1650
Wavenumber, cm
1600
-1
IR spectra of labeled Gellman A peptide:
heating from 5 (violet) to 85C (red), step
5C
Tyr
Leu
H
N
Gln
0.0
O
O
N
H
Val
N
H
H
N
O
O
Glu
Lys
H
N
Ile
O
O
N
H
Val
N
H
O
H
N
O
O
NH
H
N
Orn
Lys
O
labeled on Val3 and Lys8
Setnicka, et al. unpublished
VCD of DNA, vary A-T to G-C ratio
base deformations
sym PO2- stretches
-1
big variation
little effect
DNA VCD of PO2- modes in B- to Z-form transition
B, A
B
Z
Z
A
Experimental
B
Theoretical
Triplex DNA, RNA form by adding third strand
to major groove with Hoogsteen base pairing
VCD of Triplex formation—base modes
CGC+
-20
Wavenumber (cm-1)
• That is all for now
• Good luck on exams
• I enjoyed having you in class this Fall
• Tim Keiderling