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

hn = DE = 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.

l

E || z

B || x

n = c/l x

z

y

Wavelength Frequency

Intensity

I ~ |E|2

} Polarization

B | E

k || y

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

E = hn, where n = c/l (in sec-1)

• Intensity increases the transition probability—

I ~ e2 –where e is the radiation Electric Field strength

Linear Polarization (absorption) aligns with direction of dipole change—(scattering to the polarizability) I ~ [dm/dQ]2 where Q is the coordinate of the motion

Circular Polarization results from an interference:

Im(m • m) m and m are electric and magnetic dipole

0

.4

.8

1.2

4000 3000 2000 1000

Frequency (cm )

Abs

orba

nce

-1

l n

Intensity

(Absorbance) IR of

vegetable

oil

Optical Spectroscopy - Processes Monitored UV/ 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: DE = hn0-hns

Infrared: DE = hnvib

= hnvib

Fluorescence

hn = Eex - Egrd

0

Absorption

hn = Egrd - Eex

Excited

State

(distorted

geometry)

Ground

State (equil.

geom.)

Q

n0 nS

molec. coord.

UV-vis absorp.

& Fluorescence. move e- (change

electronic state)

high freq., intense

Analytical Methods Diatomic 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 e (M

-1 c

m-1

)

What do you see?

(typical protein)

Amide absorption broad,

Intense, featureless, far UV

~200 nm and below

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

CD is polarized differential absorption

DA = AL - AR

only non-zero for chiral molecules

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

Peptide/ Protein -

in uv - for amide: n-p* or p-p* in -HN-C=O-

partially delocalized p-system senses structure

in IR - amide centered vibrations most important

Nucleic Acids – base p-p* in uv, PO2-, C=O in IR

Coupled transitions between amides along chain lead to

distinctive bandshapes

Circular Dichroism

UV-vis Circular Dichroism Spectrometer

JASCO–quartz prisms disperse and linearly polarize light

Xe arc

source

Double prism

Monochromator (inc. dispersion,

dec. scatter, important in uv)

PEM

quartz

PMT

Sample Slits

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

Amino Acids - linked by Peptide bonds

coupling yields structure sensitivity

Link is mostly planar and trans, except for Xxx-Pro

UV absorption of peptides is featureless --except aromatics

TrpZip peptide in water

Rong Huang, unpublished

Trp – aromatic bands Amide

p-p* and n-p*

a-helix - common peptide secondary structure

(ii+4)

b-sheet cross-strand H-bonding

Anti-parallel b-sheet (extended strands)

poly-L-glu(a,____), poly-L-(lys-leu)(b,- - - -), L-ala2-gly2(turn, . . . . . )

Polypeptide Circular Dichroism ordered secondary structure types

De

l

Critical issue in CD structure studies is SHAPE of the De pattern

a-helix

b-sheet

turn

Brahms et al. PNAS, 1977

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

)baabTc

mmn

2

πR

Dipole coupling

results in a

derivative shaped

circular dichroism

De

eL-eR l

B-DNA Right -hand

Z-DNA Left-hand

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

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

Protein Circular Dichroism

Myoglobin-high helix (_______), Immunoglobin high sheet (_______)

Lysozyme, a+b (_______), Casein, “unordered” (_______),

DA

Coupling shapes, but not isolated & modeling tough

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 components

Problem – 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 De with FC helix

FC helix [%]

0 20 40 60 80

De

at

222nm

/193 n

m

0

10

(222 nm) vs FC helix

(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 a-helix,

-problematic for b-sheet or turns - solubility and stability

-old method:Greenfield - Fasman --poly-L-lysine, vary pH

i = aifa +bifb + cifc

--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

Electronic CD spectra consistent with predicted

helix content

- 3 0 0 0 0

- 2 0 0 0 0

- 1 0 0 0 0

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

1 9 02 0 02 1 02 2 02 3 02 4 02 5 02 6 0

Mo

lar

Ell

ipti

city

Wavelength (nm)

190 210 230

Note helical bands, coil has residual at 222 nm, growth of 200 nm band

Electronic 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”

6 b b sheet

, 2 )

Tyr97

Tyr25

Tyr92

H1

H3 H2

Tyr76

Tyr115

Tyr73

• 124 amino acid residues, 1 domain, MW= 13.7 KDa

• 3 a-helices

• 6 b-strands in an AP b-sheet

• 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2 trans)

Ribonuclease A

combined uv-CD

and FTIR study

Wavelength (nm)

260 280 300 320

Ellip

ticity

(mde

g)

-16

-14

-12

-10

-8

-6

-4

-2

0

Near-UV CD

Wavenumber (cm-1)

1600162016401660168017001720

Abso

rban

ce

0.00

0.01

0.02

0.03

0.04

0.05

0.06

FTIR

Wavelength (nm)

190 200 210 220 230 240 250

Ellip

ticity

(mde

g)

-15

-10

-5

0

5

Far-UV CD

Temperature 10-70oC

FTIR—amide I

Loss of b-sheet

RibonucleaseA

Far-uv CD Loss of a-helix

Near –uv CD Loss of tertiary

structure

Spectral Change

Stelea, et al. Prot. Sci. 2001

C i1 (x

102 )

-8.0

-7.6

-7.2

-6.8

-6.4

C i2 (x

10)

-1.0

-0.5

0.0

0.5

1.0

FTIRC i1

-17

-15

-13

-11

-9

-7

-5

C i2

-15

-10

-5

0

5

10

Near-UV CD

Temperature (oC)

0 20 40 60 80 100

C i1

-13

-12

-11

-10

C i2

-30

-25

-20

-15

-10

-5

0

5

Far-UV CD

Ribonuclease A

PC/FA loadings

Temp. variation

FTIR (a,b)

Near-uv CD

(tertiary)

Far-uv CD

(a-helix)

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

Temperature 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 b-sheet proteins

TFE and MeOH behave differently

thermal stability key to differentiating states

indicates residual partial order

3D Structure of Concanavalin A

Dimer (acidic, pH<6) Tetramer (pH=6-7)

Trp40

Trp88

Trp109

Trp182

High b-sheet structure, flat back extended, curved front

Monomer only at very low pH, 4 Trp give fluorescence

Effect of TFE (50%) on Con A in Far and Near UV- CD

Helical Content

pH=7 43%

pH=2 57%

Far UV-CD Near UV-CD

Helix induced with

TFE addition

Tertiary change

with TFE - loosen

Xu&Keiderling, Biochemistry 2005

Dynamics--Scheme of Stopped-flow System

Denatured

protein

solution

Refolding

buffer

solution

- add dynamics to experiment

Stopped-Flow CD for Con A Unfolding with TFE (1:1)

at Different pH Conditions

Far UV (222 nm);

[Con]f=0.2mg/ml

Near UV (290 nm);

[Con]f=2mg/ml

pH=2.0

Xu&Keiderling, Biochemistry 2005

Native state: b-sheet dominant, but high helical propensity.

Model: intramolecular ba transition pathway as opposed to folding pathways from a denatured state.

b-lactoglobulin: a protein that goes both ways!

Zhang & Keiderling, Biochemistry 2006

Lipid-induced Conformational Transition b-Lactoglobulin

1. DMPG-dependent ba transition at pH 6.8

0 1 2 3 4 5

0.1

0.2

0.3

0.4

0.5

b-Sheet

a-Helix

Unordered

Fra

ctio

nal

sec

on

dar

y s

tru

ctu

re

DMPG / mM

Zhang & Keiderling, Biochemistry 2006

Charge-induced Lipid -- b-Lactoglobulin Interaction

0 20 40 60 80 1000.1

0.2

0.3

0.4

0.5

DMPC/ (DMPC+DMPG)/ %

Unordered

Sheet

Helix

Fra

ctio

nal

sec

ondar

y s

truct

ure

DMPG / (DMPC+DMPG) / %

100 80 60 40 20 0

Increase DMPG, increases helix at expense of sheet

Zhang &

Keiderling,

Biochemistry 2006

1 Volume

Vesicles (SUV)

+

BLG (0.2mg/ml)

CD: 222nm to monitor alpha-helix

Fluorescence: filter with a 320nm cutoff ( Trp Tertiary Structure)

10-15 kinetic traces are collected and averaged

5 Volume

Vesicles (SUV)

(DOPG, DMPG, DSPG)

BLG (1.2mg/ml)

Stopped Flow Experiments : (pH 4.60)

Analysis：Multi-exponential function using Simplex Method:

S(t)=a*t+b+∑i(ci Exp(-ki*t))

Ge, Keiderling, to be submitted

Simulated kinetics traces of BLG (0.2mg/ml) with DMPG

Time/s

0 5 10 15 20

Ellip

ticit

y(m

deg

)

-50

-40

-30

-20

-10

0.15mM DMPG

0.50mM DMPG

1.00mM DMPG2.00mM DMPG5.00mM DMPG

N

0.25mM DMPG

Stopped-Flow CD kinetic traces

DMPG

Record at 222nm;

N: trace without lipid vesicles;

Traces are fitted to single-exponential function

Kinetics traces for BLG(0.2mg/ml) with DMPG

Time/s

0 5 10 15 20

Rela

tive

In

ten

sit

y

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

0.15mM DMPG

0.50mM DMPG1.00mM DMPG2.00mM DMPG5.00mM DMPG

0.25mM DMPG

Stopped-Flow fluorescence kinetics

Total fluorescence >320nm;

Each trace has been divided

by kinetic trace without lipid vesicles;

Traces are fitted to two-exponential function

DMPG

At pH 6.8 & 4.6,

4 & 6 nm blue

shift in lmax.

1700 1600 1500 1400 130016

54

1637

1745

1731

1654

1637

1467

1343

1328 13

05

1280 12

5512

29

pH 6.8

pH 4.6

Wavelength/cm-1

a-helix Membrane surface

Lipid bilayer insertion of b-Lactoglobulin

0.0 0.1 0.2 0.3 0.4

1.0

1.2

1.4

1.6

bLG

bLG-DMPG, pH4.6

bLG-DMPG, pH6.8

F0 /

F

Acrylamide/M

Zhang & Keiderling, Biochemistry 2006

ATR-FTIR orientation Fluorescence quenching

Summary: Lipid - b-Lactoglobulin Interaction

Nw Ns Unfolding Us Insertion Um Binding

Zhang & Keiderling, Biochemistry 2006

• Continued in Part b