BioMolecular Vibrational Spectroscopy, Pt. 2:

Applications of Infrared, Raman, VCD Spectra

Lectures for Warwick CD Workshop, Dec. 2011

Tim Keiderling

University of Illinois

at Chicago

tak@uic.edu

Tentative Schedule — can vary with interests

Part I: • Vibrational Spectra Theory and Practice • IR - Raman Instrumentation; techniques

Part II: • Application Examples

– Peptide bases, secondary structure types results – Avoiding aromatics – Protein differences

• Isotopes give new dimension, site specific • Dynamics and ultra fast • Imaging (no time)

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

Wavenumbers (cm-1

)

1450150015501600165017001750

Ab

so

rban

ce

0

1

2

3 helix

-structure

randomcoil

IR absorbance spectra of selected model peptides

I II

IR absorbance spectra of selected model polypeptides

(LKKL)n

helix

(LK)n

sheet

polyK

Coil (31)

H2O solution D2O solution

L=Leu K=Lys

Proteins—shift the emphasis

Multiple structural types

IR bands overlap

Deconvolve - “resolution enhance”—derivatives, FSD

Band fitting - subjective

Bandshape (Factor or Principle Component) analysis

Extra insight—

Polarization - surface sensitive experiments (ATR)

Resonance Raman – chromophore emphasis

Imaging – biological tissue, distribution

Time Domain – reactions, folding, photolysis

Circular Dichroism—chirality

Myoglobin

Hemoglobin

Cytochrome C

Citrate Synthase

-3

-2

-1

0

1

1800 1700 1600 1500 1400

Wavenumber (cm-1)

Superoxide

Dismutase

-2

-1

0

1

1800 1700 1600 1500 1400

Wavenumber (cm-1)

Lectin

Concanavalin-A

‘No’ a-helix, high -sheet High a-helix, ‘no’ -sheet

Example Protein/H2O IR spectra:

A A

FTIR and DFTIR of Lysozyme in H2O-D2O Mixtures

0

.2

.4

.6

.8

1

1750 1700 1650 1600 1550 1500 1450 1400 1350

100% H2O 100% D2O

-.4

-.2

0

.2

.4

1750 1700 1650 1600 1550 1500 1450 1400 1350

I II II’

FTIR

Inc. D2O

Difference IR

DFTIR

IRx-IRH2O

Amide I relatively small change, amide II 100 cm-1 shift, amide III more

ATR Polarization Measurements

E

IR beam multiply reflects inside crystal -- penetrates surface

keeps polarizations: Eperp in surface, Epara partially out

Sample coated

on crystal

Crystal-

Typ: Ge, ZnSe

High index for refl.

In from

FTIR

out

A ‘Few’-bounces ATR - Types available

• Often diamond over ZnSe

– Provides very hard surface

– Can apply pressure with rod above

• 1-3 bounces, signal can be small – solution difficult – Good for membranes, solids

– Easy to clean

• 9-bounce dip to hold liquid

• Design to fit your FTIR

• Several manufacturers

Structure of Bovine -lactoglobulin

Milk Protein Sequence predict: alpha helical Undergo beta to alpha transition with surfactants, lipids

Interaction between Protein and Lipid Vesicles

DMPG 14:0

-sheets a-helices

11

0 1 2 3 4 5

0.1

0.2

0.3

0.4

0.5

BpH 6.8

-Sheet

a-Helix

Unordered

Fra

ctio

nal

sec

ondar

y s

truct

ure

DMPG / mM

Secondary structure: Binding DMPG at pH6.8, causes BLG conformational change. The

a–helix formed with loss of -sheet.

2nd struct. analyses

BLG Binding to DMPG at pH 6.8: CD result

--sheet to a–helix transition, dependence on DMPG

12

X.Zhang,TAK Biochemistry 2007

Orientation of BLG into lipid membrane: -Polarized ATR-FTIR spectra of DMPG-bound BLG

3000 2900 1800 1700 1600 1500 1400 1300

1654 1637

2960

2912

2848

1731

1745 16

5416

37

1467

1343 13

2813

05 1255

1229

1280Dichroic spectrum, pH 6.8

Dichroic spectrum, pH 4.6

900 polarized spectrum

00 polarized spectrum

Wavelength / cm-1

helix sheet

CH2 str

CH2 scis

amide I

13 X.Zhang,TAK Biochemistry 2007

Time-resolved Spectroscopy Techniques

Stopped-flow CD and Fluorescence

Stopped-flow FTIR

P C

Protein

RenaturantDenaturant

Looking at the mechanism of the transition from initial to final state

Wavenumber (cm-1)

15501600165017001750

log

(S

i/S

f)

-0.03

-0.02

-0.01

0.00

0.01

0.02

1660 cm-1

loss of random coil

1630 cm-1

gain of sheet

time (s)

0 5 10 15 20

Peak In

ten

sit

y

-0.03

-0.02

-0.01

0.00

0.01

1632 cm-1

(sheet)

k = 0.156 s-1

1660 cm-1

(random coil)

k = 0.342 s-1

Refolding of Ribonuclease A by FTIR

Slow/Rapid scan FTIR - Inverse T-jump: Refolding initiated by

injecting Ribo A stored in syringe at 80 °C into IR cell at 25 °C

Sheet refolding 2x slower

than loss of coil

One single beam spectrum (IF scan) is collected for each time point.

Time res. = 50 ms. IR resolves increase coil & decrease sheet fold.

Difference IR

Heinz Fabian et al.

Concanavalin A pH, TFE and MeOH

native

TFE

MeOH normalizes -sheet ECD,

FTIR indicates aggregated

TFE induces helix Xu&Keiderling, Biochem, 2005

Stopped-flow FTIR of Con A Unfolding with TFE (1:1)

Fast Transition

(Principle Component Analysis) (Difference Spectra)

1630 cm-1

1654 cm-1

Qi Xu and TAK, Proteins

Wavenumber (cm-1)

1600165017001750

Absorb

an

ce

0.0

0.5

1.0

A

x 1

05

-10

-5

0

5

10

VCD

IR

(a)

Wavenubmer (cm-1)

1600165017001750

Ab

so

rba

nce

0.0

0.5

1.0

A x

10

5

-4

-2

0

2

IR

VCD

(b)

Poly Lysine in D2O – Amide I’–Secondary structure VCD

High pH – helix High pH, heating – sheet Neutral pH - coil

Wavenumber (cm-1)

1600165017001750

Absorb

ance

0.0

0.5

1.0

A

x 1

05

-15

-10

-5

0

5

IR

VCD

(c)

Wavenumbers (cm-1

)

140016001800

Ab

so

rba

nc

e

0

1

2

3

4

Wavenumbers (cm-1

)

140016001800

A

(A

.U.)

-100

0

100

200

300

400

500

a-helical

(Aib-Ala)6

Ala(AibAla)3

310

-helical

a-helical

310

-helical

Ala(AibAla)3

(Aib-Ala)6

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

Biphenyl bridged residues (Bip) CD and IR difficult to get structure

CD—all biphenyl Amide A shows H-bond form

Toniolo, co-workers JACS 2004

Biphenyl bridged residues (Bip) show inversion

VCD and IR spectra of Ac-(Bip)3-L-

Val-OMe (full lines) and Boc-L-Val-

(Bip)4-OtBu (dashed lines). Spectra

of Ac-(Bip)3-L-Val-OMe were

measured in 46/11 (v/v) CDCl3/TFE-

OH and Boc-L-Val-(Bip)4-OtBu in

CDCl3 solution, pathlength 500 mm

and concentration of 9.5 and 8.6 g/L,

respectively.

Ac-(Bip)3-L-Val-OMe (_________)

left-handed

Boc-L-Val-(Bip)4-OtBu (-------)

right-handed (310-helix)

Toniolo, co-workers JACS 2004

Vibrational spectrum separates

aromatic and amide transitions

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 to 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

Dukor, Keiderling - Biopoly 1991

Relationship to “random coil” - compare Pron and Glun

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

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 Aib, Ala and Aib-

Ala alternating sequences.

(Kubelka,Silva, Keiderling JACS 2002)

Simulation: a-helix

Experiment: Simulation: 310-helix

Wavenumber [cm-1

]

150016001700

/a

mid

e

Ac-(Aib)8-NH

2

Ac-(Aib-Ala)4-NH

2

Ac-(Ala)8-NH

2

Wavenumber [cm-1

]

150016001700

/a

mid

e

Ac-(Aib-Ala)3-NH

2

Ac-(Ala)6-NH

2

Simulation of Helix IR and VCD Really Works!

13C Isotopic Labeling

Change 12C to 13C on amide C=O

shift amide I down by ~40 cm-1 (isotopic shift) (13C=18O even more)

Isotopic advantage: site-specific

(specific, local Amide I vibrational coupling)

Amide I (1700-1600 cm-1)

coupling structure

IR limitation: average secondary structure

(delocalization of Amide I vibrational coupling)

Isotopes can break that - give specific sites

Basic principle of spectra-structure relationships:

Wavenumber [cm-1

]

1550160016501700

An

orm

(x 1

0)

0

4

8

12Unlabeled

N-terminus

C-terminus

Middle (N)

Middle (C)

165017001750

x 1

0-3

)

2

4

165017001750

Wavenumber [cm-1

]

1550160016501700

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 & experimental amide I IR : Ala20 - 13C labels

a-helix ProII-like

Low T High T

Silva, Kubleka, et al. PNAS 2000

Simul.

Exper.

165017001750

Unlabeled

N-terminus

C-terminus

Middle (N)

Middle (C)

165017001750

x 1

0)

-8

-6

-4

-2

0

2

Wavenumber [cm-1

]

1550160016501700

A

no

rm (

x 1

05)

-8

-4

0

4

Unlabeled

N-terminus

C-terminus

Middle (N)

Middle (C)

Wavenumber [cm-1

]

1550160016501700

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 & experimental VCD : Ala20 - 13C labels

Silva, Kubleka, et al. PNAS 2000

Temperature [oC]

10 20 30 40 50 60

Fre

quency [cm

-1]

1643

1645

1647

1649

1651

1653C-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 – identify location with isotope

Center Termini

Relative isotope position-- experiment and theory

Two sequential labels have higher IR freq. due to coupling (intensity in high n mode), VCD : sequential (2LT) - same sign 12C and 13C, but opposite sign if separated (2L1S)

* since exp. in D2O a (-)band develops the amide I, but not modeled without solvent

IR VCD

*

13C

13C

13C

13C

Huang, et al. JACS 2004

. . .AKAAAAK. . .

. . .AKAAAAK. . .

. . .AKAAAAK. . .

. . .AKAAAAK. . .

Coupling is source of variation

N C

Wavenumber [cm-1]

Simulations for antiparallel -sheet - strands offset by one residue

Wavenumber [cm-1

]

1600164016801720

Absorb

ance

*

*

*

Kubelka, Huang unpublished, 2004 - best fit!

Simulation

L34

G38

V39

Hairpin – simplest -sheet model Monomeric model - anti-parallel interaction Interpretive challenge — heterogeneous Turn very different from strands Strands are non-uniformly twisted Ends are frayed Stabilize hairpin – two methods Turn residues that lock in limited conformation

--DPro-Gly (isotope interference, Hilario, et al. JACS 2003), --Aib-Gly (Setnicka, et al. JACS 2005, Huang, et al. JACS 2007)

Chain residues interact (hydrophobic, charges) --Trp-Zip models (Cochran, PNAS 2001, many followups), --aromatic coupling (Waters, others)

heating from 5 to 85°C, step 5°C

TrpZipper

AsnNH

NH

NH

NH

NH

NH3

+

Ser

O Trp

NH

NH

NH

NH

NH

NH

Lys O O

OO

O

O

O

O

OO

Thr

Trp

Glu

Trp

Thr

Trp

Lys

NH2

O

-O

Stable hairpin – cross-strand hydrophobic interaction

Trp-Trp interactions stabilize hairpin - Cochran et al. PNAS 2000

Trp interfere in CD – provides no secondary structure information Effectively tertiary

5 C

85 C

ECD intensity change vs temperature at different pH

Temperature (K)

280 300 320 340 360

[ ] x

10

- 5 (d

eg

cm

2 d

mo

l-1

)

-8

-4

0

4

8

12

pH=6.94 at 226.8nm

pH=2.38 at 227nm

pH=6.94 at 212.8nm

pH=2.38 at 212.6nm

c

Reversable, 215, 230 nm same transition

Secondary Structure Change with

Temperature - Monitor with IR

Thermal transition from -hairpin

(low temp, 1625-30 cm-1 max.) to

disordered (high temp, 1645-50

cm-1 max.)

TZ2C intensity change at acidic pH

Temperature (K)

260 280 300 320 340 360 380A

bs

orb

an

ce .20

.25

.30

.35

.40

.45

.50

.55

c

intensity change at 1632.2cm-1

intensity change at 1652.5cm-1

Tm=321.1KTm=332.8K ____

IR intensity change at 1632 cm-1 (-

sheet, black), 1652 cm-1, (disordered,

red), fit to a two state model - transition

temperatures of 321 K and 333 K

not really two-state!! Huang, Wu, et al. JPhysChemB 2009

13C=O isotopic labeling of TZ2C

Simulation

Experiment H3N+

HN

NH

HN

NH

H2N

HN

NH

HN

NH

O O

HN

O

O

OO

Ala

Lys

Ala

Ala

Glu

Lys

HN

Trp

Trp

Trp

Trp

O

O

O

O O

NH

O

Asn

H3N+

HN

NH

HN

NH

H2N

HN

NH

HN

NH

O O

HN

O

O

OO

Ala

Lys

Ala

Ala

Glu

Lys

HN

Trp

Trp

Trp

Trp

O

O

O

O O

NH

O

Asn

H3N+

HN

NH

HN

NH

H2N

HN

NH

HN

NH

O O

HN

O

O

OO

Ala

Lys

Ala

Ala

Glu

Lys

HN

Trp

Trp

Trp

Trp

O

O

O

O O

NH

O

Asn

Cross-strand vibrational coupling is

position sensitive

A1A10

A3A10

A3K8

13C

13C

Simulations predict 13C=O coupling

DFT-level FF, NMR structure

Huang, Wu et al. JPhysChem B 2009

Summary – equilibrium variation Tm values differ for 12C and 13C modes Also differ for frequency shift and intensity change Inconsistent with 2-state model for unfolding Fits earlier conclusions, folding multistate ensemble

Dynamics might give mechanistic insight

Trpzip-based beta hairpin temperature jump IR studies enhanced by site-specific

isotope labeling

with Karin Hauser group

University of Konstanz

Previously included: Carsten Kretjschi1, Rong Huang2, Ling Wu2, Tim Keiderling2

1Institute of Biophysics, University of Frankfurt,

2Department of Chemistry, University of Illinois at Chicago

Or: why I am in Konstanz as Humbolt Fellow

5-8 µm

Nd:YAG Laser

1064 nm, 700 mJ, 10 ns

IR Laser Module

Sample

IR

Detector

1906 nm

Raman-Shifter

Tunable semiconductor lasers as IR source:

• amide I single wavelength transients

• spectral range: 2300 -1200 cm-1

• modes every 2 - 4 cm-1

• cw power up to 1 mW

• high S/R

Laser-Induced Temperature Jump &

Single Wavelength Detection

• Conformational relaxation dynamics in the ns-µs time range

• Excitation of water overtone/combination vibration at 1906 nm

40 °C; t = 1.1 ± 0.1 µs

34 °C; t = 1.8 ± 0.1 µs

27 °C; t = 2.4 ± 0.1 µs

21 °C; t = 3.5 ± 0.2 µs

15 °C; t = 5.8 ± 0.5 µs

52 °C; t = 0.6 ± 0.1 µs

46 °C; t = 0.8 ± 0.1 µs

Unfolding Dynamics of TZ2C

• relaxation kinetics at

initial temperatures

• single exponential fit:

A(t) = B exp(-t/t)

1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600

-0.20

-0.15

-0.10

-0.05

0.00

0.05

85°C

5°C

5°C

85°C

A /

OD

wavenumber / cm-1

-hairpin disordered

24 °C; t = 2.9 ± 0.2 µs

30 °C; t = 2.2 ± 0.1 µs

36 °C; t = 1.7 ± 0.1 µs

42 °C; t = 1.3 ± 0.1 µs

11 °C; t = 5.6 ± 1.2 µs

18 °C; t = 4.5 ± 0.4 µs

-loss faster Hauser, et al. JACS 2008

T-variation

Arrhenius

b

c

A1A10

A3A10 A3K8

Dynamics Labeled TZ2C 13C=O dynamics picks out

character of region

Blue—-strand Red – disordered Green – 13C=O relaxation

Hauser, et al. JACS 2008

>C=O H-N<

Two β-forms of P-L-GA and their influence on FT-IR – follows earlier work with insulin fibril formation

>C=O H-N<

H-O-C=O

1680 1660 1640 1620 1600 1680 1660 1640 1620 1600

18 cm-1 A

bw

sorb

ance

, a. u

.

Wavenumber, cm-1

1

0

50

Aggregating, self-assembled peptides (proteins)

Collaboration with Wojciech Dzwolak, Univ. Warsaw

1 form ……. 2 form ___ bifurcated H-bond

normal structure

Low pH, incubate, heat

52

TEM

SEM

Twisted fibril formation with pure enantiomers

L D L+D Fulara et al. J Phys Chem B 2011

1800 1750 1700 1650 1600 1550 1800 1750 1700 1650 1600 1550

2

1

-1

0

VCD Amplification of long range order

2L+D

L

53

2

0

2

4

1800 1750 1700 1650 1600 1550 1750 1700 1650 1600 1550

3L +D 2L +D 1.5L+D 1.25L+D

Chiral titration of L-Glu with D-Glu – only small change in IR

Fulara et al. J Phys Chem B 2011

-COOH

Acknowledgments

UIC

Helices Gangani Silva (now U.Cincinnati)

Rong Huang (now pd. Cincinnati)

Hairpins and sheets Ling Wu, (now pd Mich.State)

Heng Chi, Ahmed Lakhani

Theory

Jan Kubelka, (now U.Wyoming)

Joohyun Kim, (now LSU comp.)

Petr Bour, Acad. Sci. Prague

Anjan Roy

Vesicle-Protein - Xiuqi Zhang,

Ning Ge, Ge Zhang, Weiying Zhu

NMR Dan McElheny – UIC

T-jump IR Prof. Karin Hauser

Carsten Krejtschi

Frankfurt & Konstanz

Aib-Gly Hairpins Prof. Robert Hammer

Marcus Etienne, LSU

Aggregating peptides Wojciech Dzwolak - Warsaw

Funding: NSF, Guggenheim and Humboldt Foundation 54