Continued from part a Characteristic Amide Vibrations A often - - PowerPoint PPT Presentation

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continued from part a characteristic amide vibrations
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Continued from part a Characteristic Amide Vibrations A often - - PowerPoint PPT Presentation

Oct 12, 2022 170 likes •544 views

Continued from part a Characteristic Amide Vibrations A often obscured ~3300 cm -1 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

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SLIDE 1

Continued from part a

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SLIDE 2

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 obscured by 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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SLIDE 3

Peptide conformation depends on f, y angles

Far UV absorbance broad, little fluorescence—coupling impact small

Detection requires method sensitive to amide coupling 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

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SLIDE 4

Wavenumbers (cm-1)

1450 1500 1550 1600 1650 1700 1750

Absorbance

1 2 3 helix -structure random coil

I II

Model polypeptide IR absorbance spectra - Amide I and II

(weak IR but strong in Raman)

(Not in Raman)

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SLIDE 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 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

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SLIDE 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

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SLIDE 7

G C F M2 S M1 PEM P SC L D

D Pre- Amp Dynamic Normalization Tuned Filter  Lock-in C Lock-in Chopper ref. C PEM ref. M Transmission Feedback Lock-in A/D Interface Computer Interface Monochromator

UIC Dispersive VCD Schematic

Electronics Optics and Sampling

Yes it still exists and measures VCD!

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SLIDE 8

UIC FT-VCD

Schematic

(designed for magnetic VCD commercial

  • nes simpler)

Electronics Optics FTIR

Separate VCD Bench

Polarizer PEM (ZnSe) Sample Detector (MCT)

Optional magnet

lock-in amp filter PEM ref detector FT-computer

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SLIDE 9

Large electric dipole transitions can couple over longer ranges to sense extended conformation

Simplest representation is coupled oscillator

Tab ma mb

Real systems - more complex interactions

  • but pattern is often consistent

 )

b a ab

T c m m              

2 π R

Dipole coupling results in a derivative shaped circular dichroism

De  eL-eR l

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SLIDE 10

Selected model Peptide VCD, aqueous solution

Wavenumbers (cm-1)

1450 1500 1550 1600 1650 1700 1750

VCD (A. U.)

  • 10

10 20 30 helix -structure random coil

Amide I Amide II a  coil DA

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SLIDE 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 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

  • ligopeptides, and transient forms
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SLIDE 12

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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SLIDE 13

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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SLIDE 14

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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SLIDE 15

A

1500 1550 1600 1650 1700

HEM LYS CON

DA CON

2

Wavenumbers (cm-1) 1500 1550 1600 1650 1700 HEM LYS

VCD in H2O FTIR in H2O

Wavenumbers (cm-1)

Comparison of Protein VCD and IR

a  a/

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SLIDE 16

VCD Example: a- 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

a-Helix 310-Helix

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SLIDE 17

Wavenumbers (cm-1) 1400 1600 1800 Absorbance 1 2 3 4 Wavenumbers (cm-1) 1400 1600 1800 DA (A.U.)

  • 100

100 200 300 400 500

a-helical (Aib-Ala)6 Ala(AibAla)3 310-helical a A

The VCD success example: 310-helix vs. a-helix

Relative shapes of multiple bands distinguish these similar helices

Aib2LeuAib5 (Met2Leu)6

a 310 mixed i->i+3 i->i+4

Silva et al. Biopolymers 2002

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SLIDE 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

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SLIDE 19

Transfer of FF, APT and AAT (e.g. Ala7 to Ala20)

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

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SLIDE 20

e

1 2 3 3

a d

De

  • 4
  • 2

2 2

a d

e

1 2 3

a d

De

  • 4
  • 2

2

a d

De

Wavenumber (cm-1)

1200 1300 1400 1500 1600 1700 1800 1200 1300 1400 1500 1600 1700 1800

Uniform long helicescharacteristic, narrow bands vacuum D2O

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

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SLIDE 21

in CDCl in TFE

(Aib-Ala)

4

Wavenumber [cm

  • 1 ]

1500 1600 1700

Aib

5

  • Leu-Aib 2

(Met

2

  • Leu)

8

310-helix vs. a-helix: comparison of Aibn, Alan and (Aib-Ala)n sequences.

Simulation: a-helix Experiment: Simulation: 310-helix

Wavenumber [cm

  • 1]

1500 1600 1700

De/amide

Ac-(Aib)8-NH2 Ac-(Aib-Ala)4-NH2 Ac-(Ala)8-NH2

Wavenumber [cm

  • 1]

1500 1600 1700

De/amide

Ac-(Aib-Ala)3-NH2 Ac-(Ala)6-NH2

Simulation of Helix IR and VCD Really Works!

(Kubelka,Silva, Keiderling JACS 2002)

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SLIDE 22

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?

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SLIDE 23

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

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SLIDE 24

Wavenumber [cm-1]

1550 1600 1650 1700

Anorm (x 10)

4 8 12

Unlabeled N-terminus C-terminus Middle (N) Middle (C)

1650 1700 1750

e x 10-3)

2 4

1650 1700 1750

Wavenumber [cm-1]

1550 1600 1650 1700

Unalbeled N-terminus C-terminus Middle (N) Middle (C) Unlabeled N-terminus C-terminus Middle (N) Middle (C) Unlabeled N-terminus C-terminus Middle (N) Middle (C)

Simulated and experimental IR absorption for Ala20 with 13C labels C-term is different, do not know structure from IR a-helix ProII-like Low T High T

Silva, Kubleka, et al. PNAS 2000

Simul. Exper.

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SLIDE 25

1650 1700 1750

Unlabeled N-terminus C-terminus Middle (N) Middle (C)

1650 1700 1750

De x 10)

  • 8
  • 6
  • 4
  • 2

2

Wavenumber [cm-1]

1550 1600 1650 1700

DAnorm (x 10

5)

  • 8
  • 4

4

Unlabeled N-terminus C-terminus Middle (N) Middle (C)

Wavenumber [cm-1]

1550 1600 1650 1700

Unlabeled N-terminus C-terminus Middle (N) Middle (C) Unlabeled N-terminus C-terminus Middle (N) Middle (C)

a-helix ProII-like Low T High 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

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SLIDE 26

DA (x105)

  • 8
  • 4

4 1580 1620 1660

DA (x105)

  • 8
  • 4

4 1580 1620 1660

5 deg 10 deg 15 deg 20 deg 25 deg 30 deg 35 deg 40 deg 45 deg 50 deg 55 deg 60 deg

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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SLIDE 27

Temperature [oC]

10 20 30 40 50 60

Frequency [cm-1]

1643 1645 1647 1649 1651 1653

C-terminus N-terminus Unlabeled

Temperature [oC]

10 20 30 40 50 60

Unlabeled Middle (C) Middle (N)

a b

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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SLIDE 28

small H- bonding ring large H-bonding ring

Monomeric -sheet models – hairpins

13C=O labeling - sense cross-strand coupling

Setnicka et al. JACS 2005

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SLIDE 29

Two labeling types, distinct cross-strand coupling

Simulation Experiment

Setnicka et al. JACS 2005

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SLIDE 30

IR spectra of labeled Gellman A peptide: heating from 5 (violet) to 85C (red), step 5C

Wavenumber, cm-1 1600 1650 1700 0.0 0.2 0.4 0.6 A

labeled on Val3 and Lys8

N H N H N H N H N H N H3

+

Arg O Tyr NH N H N H N H N H N H Gln O O O O O O O O O O Val Glu Val Leu Ile Lys Orn N H2 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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SLIDE 31
  • 1

VCD of DNA, vary A-T to G-C ratio

base deformations sym PO2

  • stretches

big variation little effect

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SLIDE 32

A B

DNA VCD of PO2

  • modes in B- to Z-form transition

Experimental Theoretical Z B B, A Z

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SLIDE 33

Triplex DNA, RNA form by adding third strand

to major groove with Hoogsteen base pairing

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SLIDE 34
  • 20

CGC+ Wavenumber (cm-1)

VCD of Triplex formation—base modes

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