Continued from part a

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

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

I

II

Model polypeptide IR absorbance spectra - Amide I and II

(weak IR but strong in Raman)

(Not in Raman)

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

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

 

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!

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

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

Selected model Peptide VCD, aqueous solution

Amide IAmide II

coil

A

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

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

Dukor, Keiderling - Biopoly 1991

Relationship to “random coil” - compare Pron and Glun

IR ~ same, VCD - same shape, half size -- partially ordered

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

VCD in H2OFTIR in H2O

Wavenumbers (cm-1)

Comparison of Protein VCD and IR

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

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

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

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

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

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)

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?

-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

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.

-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

Wavenumber [cm-1]

a b

c d

Temperature dependent Ala20 VCD: a) unlabeled b) C-terminus c) N-terminus d) Middle(N) labeled

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

small H- bonding ring large H-bonding ring

Monomeric -sheet models – hairpins13C=O labeling - sense cross-strand coupling

Setnicka et al. JACS 2005

Two labeling types, distinct cross-strand coupling

Simulation Experiment

Setnicka et al. JACS 2005

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

-1

VCD of DNA, vary A-T to G-C ratiobase deformations sym PO2

- stretches

big variation little effect

A B

DNA VCD of PO2- modes in B- to Z-form transition

Experimental Theoretical

Z

B B, A

Z

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

-20CGC+

Wavenumber (cm-1)

VCD of Triplex formation—base modes

• That is all for now

• Good luck on exams

• I enjoyed having you in class this Fall

• Tim Keiderling