Electronic Spectroscopy Chem 344 final lecture topics Time out - - PowerPoint PPT Presentation

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

.4 .8 1.2 4000 3000 2000 1000

Frequency (cm ) Absorbance

• 1

l n

Intensity (Absorbance) IR of vegetable

• il

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 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

Opt ptica ical l Spe pectrosc troscopy

• py – Ele

lectronic, tronic, Examp ample le Abs bsorpti

• rption
• n an

and d Flu luor

• res

escen cence ce

Intrinsic fluorophores

• eg. Trp, Tyr

Change with tertiary structure, compactness

e (M-1 cm-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

• nly 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

(ii+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

• rdered 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

 )

b a ab

T c m m n             



2 π R

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 [%]

20 40 60 80

De at 222nm/193 nm

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
• 2
• 1

1 2 3 4 5 1 9 2 2 1 2 2 2 3 2 4 2 5 2 6

Ellipticity 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

Ellipticity (mdeg)

• 16
• 14
• 12
• 10
• 8
• 6
• 4
• 2

Near-UV CD

Wavenumber (cm-1)

1600 1620 1640 1660 1680 1700 1720

Absorbance

0.00 0.01 0.02 0.03 0.04 0.05 0.06

FTIR

Wavelength (nm)

190 200 210 220 230 240 250

Ellipticity (mdeg)

• 15
• 10
• 5

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

Ci1 (x10

2)

• 8.0
• 7.6
• 7.2
• 6.8
• 6.4
• 1.0
• 0.5

0.0 0.5 1.0

FTIR

Ci1

• 17
• 15
• 13
• 11
• 9
• 7
• 5

Ci2

• 15
• 10
• 5

5 10

Near-UV CD

20 40 60 80 100

Ci1

• 13
• 12
• 11
• 10

Ci2

• 30
• 25
• 20
• 15
• 10
• 5

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 ba 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 ba transition at pH 6.8

1 2 3 4 5 0.1 0.2 0.3 0.4 0.5

b-Sheet a-Helix

Unordered

Fractional secondary structure

DMPG / mM

Zhang & Keiderling, Biochemistry 2006

Charge-induced Lipid -- b-Lactoglobulin Interaction

20 40 60 80 100

0.1 0.2 0.3 0.4 0.5

DMPC/ (DMPC+DMPG)/ %

Unordered Sheet Helix

Fractional secondary structure

DMPG / (DMPC+DMPG) / %

100 80 60 40 20

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

5 10 15 20

• 50
• 40
• 30
• 20
• 10

0.15m 0.50m 1.00m 2.00m 5.00m N 0.25m

Sto topped pped-Flo Flow w CD ki kinetic netic tr traces ces

DM DMPG

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

5 10 15 20 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

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

St Stopped pped-Flo Flow w fl fluoresc

• rescence

ence ki kinetics netics

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

DM DMPG

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

1700 1600 1500 1400 1300

1654 1637 1745 1731 1654 1637 1467 1343 1328 1305 1280 1255 1229

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