Electronic Spectroscopy

Chem 344 final lecture topics

Time out—states and transitions

Spectroscopy—transitions between energy states of a molecule excited by absorption or emission of a photon

hν = ∆E = Ei - Ef

Energy levels due to interactions between parts of molecule (atoms, electrons and nucleii) as described by quantum mechanics, and are

characteristic of components involved, i.e. electron distributions (orbitals), bond strengths and types plus molecular geometries and atomic masses involved

Spectroscopy• Study of the consequences of the interaction of

electromagnetic radiation (light) with molecules.• Light beam characteristics - wavelength

(frequency), intensity, polarization - determine types of transitions and information accessed.

λ

E || zB || x

ν = c/λx

z

y

Wavelength Frequency

IntensityI ~ |E|2

}PolarizationB | E

k || y

Properties of light – probes of structure• Frequency matches change in energy, type of motion

E = hν, where ν = c/λ (in sec-1)• Intensity increases the transition probability—

I ~ ε2 –where ε is the radiation Electric Field strength Linear Polarization (absorption) aligns with direction of

dipole change—(scattering to the polarizability)I ~ [δµ/δQ]2 where Q is the coordinate of the motion

Circular Polarization results from an interference:Im(µ • m) µ and m are electric and magnetic dipole

0

.4

.8

1.2

4000 3000 2000 1000 Frequency (cm )

Abs

orba

nce

-1

λν

Intensity(Absorbance) IR of

vegetable oil

Optical Spectroscopy - Processes MonitoredUV/ Fluorescence/ IR/ Raman/ Circular Dichroism

IR – move nuclei low freq. & inten.

Raman –nuclei, inelastic scatter very low intensity

CD – circ. polarized absorption, UV or IR

Raman: ∆E = hν0-hνs

Infrared: ∆E = hνvib

= hνvib

Fluorescencehν = Eex - Egrd

0

Absorptionhν = Egrd - Eex

Excited State (distorted geometry)

Ground State (equil. geom.)

Q

ν0 νS

molec. coord.

UV-vis absorp.& Fluorescence.move e- (change electronic state) high freq., intense

Analytical MethodsDiatomic Model

Essentially a probe technique sensing changes in the local environment of fluorophores

Optical Spectroscopy – Electronic, Example Absorption and Fluorescence

Intrinsic fluorophores

eg. Trp, Tyr

Change with tertiary structure, compactness ε(

M-1

cm-1

)

F lu o

r es c

e nc e

Inte

nsit y

Amide absorption broad,Intense, featureless, far UV ~200 nm and below

What do you see?(typical protein)

UV absorption of peptides is featureless --except aromatics

TrpZip peptide in waterRong Huang, unpublished

Trp – aromatic bandsAmide π−π* and n-π*

Circular Dichroism

• Most protein secondary structure studies use CD

• Method is bandshape dependent. Need a different analysis

• Transitions fully overlap, peptide models are similar but not quantitative

• Length effects left out, also solvent shifts• Comparison revert to libraries of proteins• None are pure, all mixed

Circular DichroismCD is polarized differential absorption

∆A = AL - ARonly non-zero for chiral molecules

Biopolymers are Chiral (L-amino acid, sugars, etc.)Peptide/ Protein -

in uv - for amide: n-π* or π−π* in -HN-C=O-partially delocalized π-system senses structure

in IR - amide centered vibrations most importantNucleic Acids – base π−π* in uv, PO2-, C=O in IR

Coupled transitions between amides along chain lead to distinctive bandshapes

Polarization Modulation with PEM for CD(courtesy Hinds Instr.)

UV-vis Circular Dichroism Spectrometer

Xe arcsource

Double prism Monochromator (inc. dispersion, dec. scatter, important in uv)

PEM quartz

PMT

SampleSlits

This is shown to provide a comparison to VCD and ROA instruments

JASCO–quartz prisms disperse and linearly polarize light

Polypeptide Circular Dichroismordered secondary structure types

∆ε

λ

α-helix

β-sheet

turn

Brahms et al. PNAS, 1977

poly-L-glu(α,____), poly-L-(lys-leu)(β,- − - −), L-ala2-gly2(turn, . . . . . )

Critical issue in CD structure studies is SHAPE of the ∆ε pattern

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

Simplest representation is coupled oscillator

Tabµaµb

( )baabTc µµν rrr

m ×⋅⎟⎠⎞

⎜⎝⎛=±

2πR

∆ε =

εL−εR

Dipole coupling results in a derivative shaped circular dichroism

λ

Real systems - more complex interactions- but pattern is often consistent

B-DNARight -hand

Z-DNALeft-hand

B- vs. Z-DNA, major success of CD

Sign change in near-UV CD suggested the helix changed handedness

Protein Circular Dichroism

∆A

Myoglobin-high helix (_______), Immunoglobin high sheet (_______)Lysozyme, a+b (_______), Casein, “unordered” (_______),

Simplest Analyses –Single Frequency Response

Basis in analytical chemistry Beer’s law response if isolated

Protein treated as a solution % helix, etc. is the unknown

Standard in IR and Raman, Method: deconvolve to get componentsProblem – must assign component transitions, overlap

-secondary structure components disperse freq.

Alternate: uv CD - helix correlate to negative intensity at 222 nm, CD spectra in far-UV dominated by helical contribution

Problem - limited to one factor, -interference by chromophores]

Single frequency correlation of ∆ε with FC helix

FC helix [%]

0 20 40 60 80

∆ε

at 2

22nm

/193

nm

0

10

θ(222 nm) vs FC helixθ(193 nm) vs FC helix

Problem of secondary structure definition No pure states for calibration purposes

?

?

?

?

helix

sheet

Where do segments begin and end?Need definition:

Next step - project onto model spectra –Band shape analysis

Peptides as models- fine for α-helix, -problematic for β-sheet or turns - solubility and stability-old method:Greenfield - Fasman --poly-L-lysine, vary pH

θi = aiφα +biφβ + ciφc--Modelled on multivariate analyses

Proteins as models - need to decompose spectra- structures reflect environment of protein- spectra reflect proteins used as models

Basis set (protein spectra) size and form - major issue

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 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

Reference: Poly(Lys) – “coil”, pH 7

ECD of Pron oligomers

Builds up to Poly-Pro II frequency -->tertiary amide helix

sheet

‘coil’

Single amide

Greenfield & Fasman 1969

Poly Proline mutarotate from I (right hand cis) to II (left hand trans)

Time dependent change in H2OPoly-L-Pro I

I+II

I

Conformation driven by added salt, concentrationPoly(LKKL) Poly(KL) (LKKL)5 (KL)10

Electronic CD spectra consistent with predicted helix content

- 3

- 2

- 1

0

1 0

2 0

3 0

4 0

5 0

1 92 02 2 2 2 2 2

ticity

Wavelength (nm)190 210 230

Note helical bands, coil has residual at 222 nm, growth of 200 nm bandElectronic CD for helix to coil change in a peptide

Loss of order becomes a question --ECD long range sensitivity cannot determine remaining local order

Low temp helix

High temp “coil”

BETA-LACTOGLOBULIN

• Mw 18,400 Da, 162 residues• Primarily β-sheet (42% sheet, 16% helix)• High propensity for helical conformation• Structural homolgy to retinol binding protein

wavelength (nm)180 200 220 240 260

-6

-4

-2

0

2

4

6

8∆

ε (M

-1cm

-1)

0 mM

3 mM} 5 - 50 mM

Far-UVCD spectra of BLG titrated with SDS (0-50 mM)

wavelength (nm)260 280 300 320

∆ε

(M-1

cm-1

)

-0.0020

-0.0015

-0.0010

-0.0005

0.0000

0.0005

0 mM

0.1 mM

1.0 mM

3, 5 mM

Near-UVCD spectra of BLG titrated with SDS

PC/FA determined secondary structure change

[SDS] (mM)0 10 20 30 40 50

Seco

ndar

y St

ruct

ure

(%)

10

15

20

25

30

35

40

sheet

helix

Critical Micelle Concentration (8.2 mM)

Tyr97

Tyr25

Tyr92

H1

H3H2

Tyr76

Tyr115

Tyr73Ribonuclease A combined uv-CD and FTIR study

6 β β sheet

, 2 )

• 124 amino acid residues, 1 domain, MW= 13.7 KDa• 3 α-helices• 6 β-strands in an AP β-sheet• 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2 trans)

W a v e l e n g t h ( n m )2 6 0 2 8 0 3 0 0 3 2 0

Ellip

ticity

(mde

g)

- 1 6

- 1 4

- 1 2

- 1 0

- 8

- 6

- 4

- 2

0

N e a r - U V C D

W a v e n u m b e r ( c m - 1 )1 6 0 01 6 2 01 6 4 01 6 6 01 6 8 01 7 0 01 7 2 0

Abs

orba

nce

0 . 0 0

0 . 0 1

0 . 0 2

0 . 0 3

0 . 0 4

0 . 0 5

0 . 0 6

F T I R

Ellip

ticity

(mde

g)

- 1 5

- 1 0

- 5

0

5

F a r - U V C D Temperature 10-70oC

FTIR—amide ILoss of β-sheet

RibonucleaseA

Far-uv CDLoss of α-helix

Near –uv CDLoss of tertiary structure

Spectral Change1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0

Stelea, et al. Prot. Sci. 2001W a v e l e n g t h ( n m )

Ci1

(x10

2 )

- 8 . 0

- 7 . 6

- 7 . 2

- 6 . 8

- 6 . 4

- 1 . 0

- 0 . 5

0 . 0

0 . 5

1 . 0

F T I RC

i1

- 1 7

- 1 5

- 1 3

- 1 1

- 9

- 7

- 5

Ci2

- 1 5

- 1 0

- 5

0

5

1 0

N e a r - U V C D

0 2 0 4 0 6 0 8 0 1 0 0

Ci1

- 1 3

- 1 2

- 1 1

- 1 0

Ci2

- 3 0

- 2 5

- 2 0

- 1 5

- 1 0

- 5

0

5

F a r - U V C D

Ribonuclease APC/FA loadingsTemp. variation

FTIR (α,β)

Near-uv CD(tertiary)

Far-uv CD(α-helix)

Pre-transition - far-uv CD and FTIR, not near-uvTemperature

Stelea, et al. Prot. Sci. 2001

Changing protein conformational order by organic solvent

TFE and MeOH often used to induce helix formation--sometimes thought to mimic membrane--reported that the consequent unfolding can lead to aggregation and fibril formation in selected cases

Examples presented show solvent perturbation of dominantly β-sheet proteins

TFE and MeOH behave differentlythermal stability key to differentiating statesindicates residual partial order

β-lactoglobulin--pH and TFE , MeOH

TFE and MeOH both induce helix at both pH 7 and 2

FTIR vs. time MeOH mix

Factor analysis - 2nd component loading shows loss of sheet with time, double exp.

Xu&Keiderling, Adv.Prot.Chem.2002

Concanavalin A pH, TFE and MeOH

native

TFE

MeOH normalizes β-sheet ECD, FTIR indicates aggregated

TFE induces helixXu&Keiderling, Biochem, 2005

VIBRATIONAL OPTICAL ACTIVITYVIBRATIONAL OPTICAL ACTIVITYDifferential Interaction of a Chiral Molecule with Left and Right Circularly Polarized Radiation During Vibrational ExcitationVIBRATIONAL CIRCULAR DICHROISM RAMAN OPTICAL ACTIVITYDifferential Absorption of Left and Right Differential Raman Scattering of Left Circularly Polarized Infrared Radiation and Right Incident and/or Scattered

Radiation

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 IRsame 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

G

C F

M2S

M1

PEMP

SCL

D

D Pre-Amp

DynamicNormalization

TunedFilter

ωΜLock-in

ωCLock-in

Chopper ref. ωC

PEM ref.ωM

TransmissionFeedback Lock-in

A/DInterfaceComputerInterfaceMonochromator

UIC Dispersive VCD Schematic

Electronics

Optics and Sampling

Yes it still exists and measures VCD!

UIC FT-VCDSchematic(designed for magnetic VCD commercial ones simpler)

Electronics

OpticsFTIR

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

Tabµaµb

( )baabTc µµν rrr

m ×⋅⎟⎠⎞

⎜⎝⎛=±

2πR

∆ε =

εL−εR

Dipole coupling results in a derivative shaped circular dichroism

λ

Real systems - more complex interactions- but pattern is often consistent

Selected model Peptide VCD, aqueous solution

W a v e n u m b e r s ( c m - 1 )

1 4 5 01 5 0 01 5 5 01 6 0 01 6 5 01 7 0 01 7 5 0

VCD

(A

. U.)

- 1 0

0

1 0

2 0

3 0 h e l i x

β - s t r u c t u r e

r a n d o mc o i l

Amide IAmide II

α

β

coil

∆A

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

VCD loses magnitude

IR shifts frequency

T = 5oC (___)25oC (- - -)75oC (-.-.-)

“random coil” must have local order

Keiderling. . . Dukor, Bioorg-MedChem 1999

Salt denaturation of “random coil” polypeptidesGlun/4.5 M LiClO4 Lysn/5 M NaCl

D2O D2O

LiClO4 NaCl

Keiderling. . . Dukor, Bioorg-MedChem 1999

Lysn local order as function of length

MW ~ 1000 (- - -)25,000 (-----)

250,000 (____)

More length -more structure

pH ~ 7 -- coil(local 31-helix)

pH ~11 -- helix/coil

VCD Example: α- 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

α-Helix 310-Helix

Wavenumbers (cm-1)

140016001800

Abs

orba

nce

0

1

2

3

4

Wavenumbers (cm-1)

14016001800

∆A

(A.U

.)

-100

0

100

200

300

400

500

α-helical

(Aib-Ala)6

Ala(AibAla)3

310-helical

The VCD success example: 3The VCD success example: 31010--helix vs. helix vs. αα--helixhelix

Relative shapes of multiple bands distinguish these similar helices

Aib2LeuAib5

(Met2Leu)6 α

310

mixed

i−>i+3

i−>i+4

Silva et al. Biopolymers 2002

-1

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

big variation little effect

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

A

Z

B

B

B, A

Z

Experimental Theoretical

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

Electronic SpectroscopySpectroscopyProperties of light – probes of structureOptical Spectroscopy - Processes MonitoredUV/ Fluorescence/ IR/ Raman/ Circular DichroismCircular DichroismVCD Example: - vs. the 310-Helix