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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

E = E i - E f 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.

Intensity I ~ |

| 2

z B | E E B

|| z || x } Polarization

y k

|| y

l Wavelength n = c/ l Frequency

Properties of light – probes of structure

• Frequency  matches change in energy, type of motion

E = h

n , where n = c/ l (in sec -1 ) • Intensity 

I ~

increases the transition probability — – where e is the radiation Electric Field strength Linear Polarization dipole change —(scattering to the polarizability) 

I ~ [

Q] 2

(absorption) aligns with direction of where

is the coordinate of the motion Circular Polarization 

Im(

m •

m and

results from an interference: are electric and magnetic dipole Intensity (Absorbance) 1.2

IR of vegetable oil .4

n l 0 4000 3000 2000 1000

Optical Spectroscopy - Processes Monitored UV/ Fluorescence/ IR/ Raman/ Circular Dichroism

Excited State (distorted geometry)

Diatomic Model Analytical Methods Absorption

h n = E grd - E ex

UV-vis absorp.

& Fluorescence

move e (change electronic state) high freq., intense Ground State (equil. geom.) n 0 n S

Fluorescence

h n = E ex - E grd

– circ. polarized absorption, UV or IR

Raman:

D E = h n 0 -h n s = h n vib

Raman

–nuclei, inelastic scatter very low intensity 0

Infrared:

molec. coord.

D E = h n vib

– move nuclei low freq. & inten.

Optical Spectroscopy – Electronic, Example Absorption and Fluorescence

Essentially a probe technique sensing changes in the local environment of fluorophores What do you see?

(typical protein)

Intrinsic fluorophores eg. Trp, Tyr Change with tertiary structure, compactness 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

Circular Dichroism

CD is polarized differential absorption

A = A

- A

R only non-zero for chiral molecules Biopolymers are Chiral (L-amino acid, sugars, etc.) Peptide/ Protein in uv - for amide: n-

* or

p-p

* in -HN-C=O partially delocalized

-system senses structure in IR - amide centered vibrations most important Nucleic Acids – base

p-p

* in uv, PO 2 , C=O in IR Coupled transitions between amides along chain lead to distinctive bandshapes

UV-vis Circular Dichroism Spectrometer

Sample Slits

PMT

PEM quartz Xe arc source Double prism Monochromator (inc. dispersion, dec. scatter, important in uv)

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

JASCO

– quartz prisms disperse and

linearly polarize

light

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

Amide p-p * and n p * Trp – aromatic bands TrpZip peptide in water Rong Huang, unpublished

-helix - common peptide secondary structure (i



i+4)

-sheet cross-strand H-bonding

Anti-parallel

-sheet (extended strands)

Polypeptide Circular Dichroism

ordered secondary structure types a -helix b -sheet turn Brahms et al. PNAS, 1977 l poly-L-glu( a ,

____

), poly-L-(lys-leu)( b, -), L-ala 2 -gly 2 (turn,

. . . . .

)

Critical issue in CD structure studies is SHAPE of the

pattern

Large electric dipole transitions can couple over longer ranges to sense extended conformation Simplest representation is coupled oscillator

R    π n 2



T ab

 



) m a T ab m b De  e L -e R l

Dipole coupling results in a derivative shaped circular dichroism Real systems

- more complex interactions - but pattern is often consistent

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”

( _______ )

, 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

with FC helix

 (222 nm) vs FC helix  (193 nm) vs FC helix 10 0 0 20 40 FC helix [%] 60 80

Problem of secondary structure definition No pure states for calibration purposes ?

helix ?

sheet

Need definition: Where do segments begin and end?

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 = a i f a +b i f b + c i f c - 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 for helix to coil change in a peptide Electronic CD spectra consistent with predicted

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

5 helix content 0 4 3 2 1 0

Loss of order becomes a question - determine remaining local order

0 High temp “coil” 0 0 1

190

2 1 3 2 2

230

Wavelength (nm) 2 2 2 9 0 1 0 0 0 0 0 2 0 0 0 3 4 5 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Tyr92 Tyr97 H1 Tyr115 Tyr73

Ribonuclease A combined uv-CD and FTIR study

H2 H3 Tyr76 Tyr25 •

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

•

-helices

• •

-strands in an AP

-sheet

b sheet )

5 0 -5 -10 -15 -10 -12 -14 -16 0 -2 -4 -6 -8 0.06

0.05

0.04

0.03

0.02

0.01

0.00

1720 FTIR 1700 260 1680 1660 1640 Wavenumber (cm -1 ) 1620 1600 Near-UV CD 280 300 Wavelength (nm) 320 190 200 210 220 Wavelength (nm) RibonucleaseA FTIR

—amide I Loss of b -sheet

Near –uv CD

Loss of tertiary structure

Far-uv CD

Loss of a -helix

Far-UV CD 230 240 Spectral Change Temperature 10-70 o C 250

Stelea, et al. Prot. Sci. 2001

-6.4

-6.8

-7.2

-7.6

-8.0

FTIR 1.0

Ribonuclease A 0.5

0.0

PC/FA loadings Temp. variation

-0.5

-1.0

FTIR (

a,b

) -5 -7 -9 -11 -13 -15 -17 Near-UV CD 10 5 0 -5 -10 -15 Near-uv CD (tertiary) -10 -11 -12 Far-UV CD 5 0 -5 -10 -15 ( Far-uv CD

-helix) -20 -13 -25 0

Pre-transition

20 40 60 80 100 Temperature ( o C)

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

-30

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

Trp40

Dimer

(acidic, pH<6) Trp109

Tetramer

(pH=6-7) Trp182 Trp88 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 Far UV-CD Near UV-CD Helix induced with TFE addition

pH=7 pH=2 Helical Content 43% 57%

Tertiary change with TFE - loosen

Xu&Keiderling, Biochemistry 2005

Dynamics--Scheme of Stopped-flow System

- add dynamics to experiment Denatured protein solution Refolding buffer solution

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

-lactoglobulin: a protein that goes both ways!

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

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

Zhang & Keiderling, Biochemistry 2006

Lipid-induced Conformational Transition

-Lactoglobulin

1. DMPG-dependent ba transition at pH 6.8

0.5

Unordered 0.4

0.3

a -Helix 0.2

b -Sheet 0.1

0 1 2 3 DMPG / mM 4 5 Zhang & Keiderling, Biochemistry 2006

Charge-induced Lipid --

-Lactoglobulin Interaction

0.5

100 DMPC/ (DMPC+DMPG)/ % 80 60 40 20 0 Unordered 0.4

Helix 0.3

0.2

Sheet 0.1

0 20 40 60 80 100 DMPG / (DMPC+DMPG) / % Zhang & Keiderling, Biochemistry 2006 Increase DMPG, increases helix at expense of sheet

Stopped Flow Experiments : (pH 4.60)

Vesicles (SUV) (DOPG, DMPG, DSPG ) BLG (1.2mg/ml) 5 Volume 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

Analysis

： Multi-exponential function using Simplex Method:

S(t)=a*t+b+ ∑ i (c i Exp(-k i *t))

Ge, Keiderling, to be submitted

-10 -20 -30 -40 -50 0 DMPG 5 10

Time/s

N 0.15mM DMPG 0.25mM DMPG 0.50mM DMPG

1.00mM DMPG 2.00mM DMPG 5.00mM DMPG

Record at 222nm; N: trace without lipid vesicles; Traces are fitted to single-exponential function

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

0 DMPG 5 10

Time/s

5.00mM DMPG 2.00mM DMPG 1.00mM DMPG 0.50mM DMPG 0.25mM DMPG 0.15mM DMPG

20 Total fluorescence >320nm; Each trace has been divided by kinetic trace without lipid vesicles; Traces are fitted to two exponential function

Lipid bilayer insertion of

-Lactoglobulin Fluorescence quenching ATR-FTIR orientation

1.6

b LG b LG-DMPG, pH4.6

b LG-DMPG, pH6.8

pH 6.8

1.4

1.2

1.0

0.0

0.1

0.2

0.3

Acrylamide/M 0.4

At pH 6.8 & 4.6, 4 & 6 nm blue shift in l max .

1700 pH 4.6

1600 1500 1400 Wavelength/cm -1 1300 a

-helix



Membrane surface

Zhang & Keiderling, Biochemistry 2006

Summary:

Lipid -

-Lactoglobulin Interaction N w

Binding

N s

Unfolding

U s

Insertion

U m

Zhang & Keiderling, Biochemistry 2006

• Continued in Part b

Slide 1

Transcript Slide 1

Electronic Spectroscopy

Time out—states and transitions

Spectroscopy

Properties of light – probes of structure

Circular Dichroism

Circular Dichroism

A = A

- A

UV-vis Circular Dichroism Spectrometer

Amino Acids - linked by Peptide bonds

coupling yields structure sensitivity

-sheet cross-strand H-bonding

Anti-parallel

-sheet (extended strands)

Polypeptide Circular Dichroism

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

Protein Circular Dichroism

Simplest Analyses – Single Frequency Response

Next step - project onto model spectra –Band shape analysis

Ribonuclease A combined uv-CD and FTIR study

Summary:

Directory