Lecture 22

Fluorescence anisotropyAmide vibrations and conformation

Time-resolved polarizationCircular dichroism

NC State University

Chemistry 431

Fluorescenceanisotropy

Fluorescence occurs after vibrational relaxation

1. Excitation2. Vibrational

relaxation3. Fluorescence4. Ground state

vibrationalrelaxation

Fluorescenceand

Competing Processes

This is known as a Jablonski diagram.Different vibrationalstates with each electronic stateare shown as parallellines.

Fluorescence lifetime and quantum yield

The intrinsic lifetime for a single state is givenby a single exponential with time constant τobs:

The quantum yield is the ratio of the moleculesthat decay by the fluorescent pathway to the total:

N t = N 0 e– t/τ0

φ =k f

k f + k other= ττ0

Polarization anisotropy in emissionIf a molecule is excited using polarized lightthe polarization of the emission will change ina time-dependent fashion as the molecule rotates.

MolecularRotation

IncidentElectromagneticWave

EmittedElectromagneticWave

The transition momentrotates with the molecule

NOTE:The moleculecan rotate byany angleprior toemission.

Fluorescence anisotropy

The fluorescence anisotropy is defined as:

We consider the case of high viscosity wheremolecular rotation does not occur, r0. The anglebetween the absorbing transition dipole and theemitting transition dipole is γ.

r =I || – I⊥

I || + 2I⊥

r0 = 15

3cos2γ – 1

The role of amide bands of the peptide as conformational

markers

Background• FTIR and Raman studies of amide band

vibrations can be used to monitor the change in conformation of the peptide backbone.

• X-ray requires a protein that can be crystallized.

• NMR is limited to molecular weights less than 35 kD.

• Both require much more data analysis than FTIR spectra.

Description of the amide normal mode vibrations

• The peptide group, the structural repeat unit of the protein backbone gives up to nine characteristic bands.

• Amide A (3500 cm-1) and B (3100 cm-1) are N-H stretches.

• Amide I is predominantly C=O stretch.• Amide II is N-H bending and C-N stretching.• Amide III and IV are complex in-plane modes.• Amide V-VII are out-of-plane vibrations.

Krimm & Bandekar, Adv Protein Chem 1986;38:181-364

IR spectrum of the amide vibrations

Correlations are observed between frequency and structure

Once the correlation of a set of frequencies of band shape with α-helix, β-sheet, and random coil structures has been established, the complex line shape of a protein that contains these structures can be fit usingLorentzian andGaussian lineshapes.

Susi & BylerMethods Enzymol1986;130:290

Native LysozymeThe structure ispredominantly α-helix in the corewith some surfaceexposed β-sheetregions.The ribbon followsthe peptide back-bone of the protein.

FTIR of amide I of native lysozymeThe secondary structure prediction based on the amide I band is in agreement with the lysozyme structure.

β-sheet α-helix random β-sheet

Denatured lysozymeIt would be very difficult to determine the crystal structure of a denatured protein. the FTIR technique allows one to predict the composition of various secondary structural components in various folding states.

Structure of a β-sheet

Experiment

Calculation

β-helix model

Transitions of Elastin-like Protein

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.60.00

0.02

0.04

0.06

0.08

0.10

0.12

Abs

orba

nce

at m

axim

um

[NaCl], M

25 30 35 40 45 50 55

0.00

0.01

0.02

0.03

0.04

Abs

orba

nce

Temperature, oC

1500 1600 17000.00

0.01

0.02

0.03

Abs

orba

nce

Wavenumber, cm-1

30oC 43oC 46oC

1500 1550 1600 1650 1700

0.00

0.05

0.10

Abs

orba

nce

Wavenumber, cm-1

Infrared spectroscopy as a marker for myoglobin

dynamics

Time-resolved spectroscopyPolarization anisotropy

Myoglobin StructureGlobular α-helical protein

Heme

A helixF helix

B helixB helix

E helix

G helix

H helix

Takano, JMB, 1977, 110, 569

Ligand recombination in myoglobin

kescape

kbimolecular

His - FeP::CO

His-FeP + CO

His-FeP-CO

kgeminatehν

His-FeP*-CO

kphotolysis

kinternal

The electronic absorptionspectrum of Mb changes when CO is photolyzed.

The iron in heme is the binding site for oxygen and other diatomics

Heme is iron proto-porphyrin IX. Fe isfound in Fe2+ and Fe3+

oxidation states.Diatomics bind to Fe2+.Examples, CO, NO, O2.O2 is the physiologicallyrelevant ligand, but it canoxidize iron and it is difficultto study directly.

N N

NN

Fe

O O-O O-

O|||C

The structure of the globin changes upon deligation of carbonmonoxide

Deoxy MbMbCOThe peptide back-bone shifts and theheme changes structure when COis photolyzed. Thestructures shown areat equilibrium.

X-ray dataKuriyanTakano

CO recombination can be monitored by transient absorption spectroscopy

Excitation 532 nmProbe 432 nm

Pulsed YAG Laser ZAP!

Xe lamp

Silicon detector

MbCOSample

The spectrumchanges withtime.

PumpProbe

Ligand recombination is a sum of single exponential processes

S(t) = Φge(t)e– (k gem + k esc)t + Φbi(t)e– k bit

Difference spectrum from nano-second transient absorption spectroscopy

kescape

kbimolecular

His - FeP::CO

His-FeP + CO

His-FeP-CO

kgeminatehν

His-FeP*-CO

kphotolysis

kinternal

Myoglobin structure changes upon Fe-CO photolysis can be monitored

in the amide I bandIron-bound COCarbonyl CO of peptide

Polarization anisotropy in absorptionPhotoselection using polarized light means thatabsorptive transitions of the molecule can be used to probe molecule motions.

MolecularRotation

IncidentElectromagneticWave (PUMP)

IncidentElectromagneticWave (PROBE)

The transition moments of all electronic andvibrational transitions rotate with the molecule.

transition moment 1 transition moment 2

Polarization ratio

The polarization ratio is defined as:

For the carbonyl group of heme:

where α is the angle of C=O with respect tothe heme plane.

R = A⊥

A||

R =4 – sin2α

2 + 2 sin2α

Time-resolved infrared difference spectra of photolyzed MbCO500 ns transient obtained by step-scanin 75% glycerol/buffer solution

Franzen and Dyer, Unpublished

C=OR = 0.75a = 90o

Amide Ianalysisof ΔA isneeded!

The residues in the turn regions of Mb are most affected by the

conformational changes

R

Ψ

φ

Direction of chain

It is the change in the projection of the amide C=O dipoles on the heme plane

that results in change in anisotropy.

C=O Projection Angles Change in C=O in Angles

The major changes occur in the turns!

Ultrafast mid-infrared shows CO trapping inside the distal pocket of Mb

C O

O C

Two CO orientations observedin infrared bands B1 and B2

Spectral bands correspond torotamers of CO trapped in Mb

Nature structural BiologyLim, Jackson, Anfinrud, 1997, 4, 209

Hemoglobin: the role of the iron as the trigger for

the R - T switch

The cooperative R - T switch

Hemoglobin is composed of two α and two βsubunits whose structure s resemble myoglobin.

Eaton et al. Nature Struct. Biol. 1999, 6, 351

The frequency of the iron-histidine vibration shows strain in T state

The comparison of photolyzedHbCO in the R state andthe equilibrium T state.Hb*CO at 10 ns Fe-His = 230 cm-1

Deoxy HbFe-His = 216 cm -1The lower frequency indicatesweaker bonding interactionand coupling to bending modes.

λexc = 435 nmFe-His

Deoxy HbT-state

Hb*CO10 nsR-state

The motion of the F-helix tugs on the proximal histidine and introduces strain

The frequency lowering in the T state arisesfrom weaker Fe-His ligation and from anharmonic coupling introduced by thebent conformation of the proximal histidine.

FeN

NH

FeN

NH

R state T state

Time-resolved resonance Raman can follow the R - T structure change

Strain is introduced instages as intersubunitcontacts are made.Based on the x-ray datait was proposed that theiron displacement fromthe heme plane is a triggerfor the conformational changes.

Hb*CO

10 ns

100 ns400 ns1 μs

8 μs

15 μs40 μs60 μs

120 μs

Deoxy Hb

Time evolution

200 210 220 230 240Raman Shift (cm-1)

Scott and Friedman JACS 1984, 106, 5877

Ultrafast resonance Raman spectroscopy shows that heme

doming occurs in <1 ps

Equilibrium HbCO

Difference spectra obtainedby subtraction of the redspectrum from spectra obtained at the timedelays shown. The ultrafastiron doming indicates thatthe heme iron is the trigger.

Franzen et al. Nature Struct. Biol. 1994, 1, 233

Infrared and fluorescence spectroscopic studies

of protein folding

Protein folding studied by infrared temperature jump transient absorption

Excitation 2 μProbe 1680 cm-1

Pulsed YAG Laser ZAP!

IR laser

MCT detector

Sample

2 μ excitationexcites the bendingovertone of H2O.

Cyclic β-sheet forming peptidesshow very rapid (un)folding

Proline

Proline

Lysine

Lysine Tyrosine

Tyrosine

Hydrogen bonds

Difference FTIR spectra of cyclic hexamer as a function of temperature

-40x10-3

-30

-20

-10

0

10

20

Del

ta O

D

175017001650160015501500Wave Number (cm -1)

Temperature (C)

14.9 21.0 27.8 31.3 38.5 45.8 52.9 59.8 66.7 74.0 81.0 88.2

ΔA

Probe at 1620 cm-1

This differencespectrum showsthe unfoldingof the peptide.

Wavenumber (cm-1)

Temperature

oC

Formation of hydrogen bonds in cyclic β-sheet hexamer is rapid

The kinetics of unfolding of the hexamer is almost identicalin 50% glycerol as in buffer

Probe at» 1620 cm-1

The time scale for α-helix folding can be followed by a T-jump

fluorescence experimentTryptophan fluorescence

Eaton et al. Acc. Chem. Res. 1998, 31, 745

Heating

Helix melting

β-sheet folding observed by

T-jump Tryptophanfluorescence

Dansylfluorescence

Eaton et al. Acc. Chem. Res. 1998, 31, 745

GB1

GB1

GB1 β-sheet folding occurs in 3.5 μs

Eaton et al. Acc. Chem. Res. 1998, 31, 745

Tryptophan fluorescence is higher in the hydrophobicfolded state and decreases when exposed to water.

Protein folding studies reveal the time scale for formation of

secondary structure

Folding of α-helix ≈ 200 nsFolding of β-sheet ≈ 1-5 msFolding of cyclic β-sheet ≈ 50 ns

Folding consists of a nucleation event followedby chain propagation. The slow step in β-sheetformation is nucleation.

Vibrational spectroscopy of nucleic acids and DNA

The components of DNA• The individual nucleobases are:

• The nucleoside is formed by a bond with the ribose sugar at the N9 position of A and G and the N6 position of C and T.

N

N NH

N

NH2

HN

N NH

N

O

H2N

N

NH

NH2

O

NH

NH

O

O

Adenine Guanine Cytosine Thymine

The stability of double-helical DNA

O

HOH

HH

HH

HO

N

N

NH2

O

The double helical form of DNA is formed by phosphodiesterlinkages between the 5’ -OH end of one ribose and the 3’-OH of the next.The double helix is stabilized by hydrogen bonding in canonical base pairs and by stacking interactions between the bases.

Infrared spectra of cytosine indicate the correct tautomeric form

Even though the Watson-Crick double helical structure was known in 1953, the hyrogen bondpattern needed to be determined. Infrared spectroscopy played a keyrole as shown here.

Miles (1961)

DNA conformations• DNA is a biopolymer with three distinct

conformations these are known as B, A, and Z.

• The B form of DNA is most widely observed in fibers at low humidity and solution studies at low salt concentration.

• As the fiber humidity or solution salt concentration is increased the A form and then the Z form are observed.

DNA conformations

A form B form Z form

Conformations of the ribose

• B is 2’-endo anti• A is 3’-endo anti• Z is 2’-endo syn

The first X-ray crystal structure of d(GCGCGC) was in the Z-

form• Watson-Crick DNA was determined based on

fiber diffraction. This was B form.

• The expectation that crystalline DNA would also be in the B-form was shattered by the structure of the hexamer d(GCGCGC)

• Raman and infrared spectroscopy have played a key role in comparisons of the various forms of DNA.

Raman spectroscopic markers of A, B, and Z form DNA

600 800 600 800Raman Shift (cm-1)

protonated deuterated

Raman difference spectra of poly(dGC) at low and high salt

Wavenumber (cm-1)

Infrared spectra of DNA

AZ

A

B

B

Poly(dA.dT) fibersPoly(dGC.dCG) fibers

Circular dichroism

Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. The absence of regular structure results in zero CD intensity, while an ordered structure results in a spectrum,which can contain both positive and negative signals.

At a given wavelength,ΔA = (AL − AR)

where ΔA is the difference between absorbance of leftcircularly polarized (LCP) and right circularly polarized (RCP) light. It can also be expressed as:

ΔA = (εL − εR) C lwhere εL and εR are the molar extinction coefficients for RCP and LCP light, where C is the molar concentration and l is the path length.Then

Δε = (εL − εR)is the molar circular dichroism.

Although ΔA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity.The molar ellipticity is:

[θ] = 3298Δε.

Circular dichroism

At a given wavelength,ΔA = (AL − AR)

where ΔA is the difference between absorbance of leftcircularly polarized (LCP) and right circularly polarized (RCP) light. It can also be expressed as:

ΔA = (εL − εR) C lwhere εL and εR are the molar extinction coefficients for RCP and LCP light, where C is the molar concentration and l is the path length.Then

Δε = (εL − εR)is the molar circular dichroism.

Although ΔA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity.The molar ellipticity is:

[θ] = 3298Δε.

Circular dichroism

Circularly Polarized LightCircularly polarised light can be described in terms of electric (e) and magnetic (m) wave components. Linearly andcircularly polarized light are contrasted below.

Application to biological moleculesIn general, this phenomenon will be exhibited in absorption bands of any optically active molecule. As a consequence, circular dichroism is exhibited by biological molecules, becauseof the dextrorotary (e.g. some sugars) and levorotary (e.g. some amino acids) molecules they contain. Noteworthy as well is that secondary structure will also impart a distinct CD to their respective molecules. Therefore, the alpha helix of proteins and the double helix of DNA have CD spectral signatures representative of their structures.The ultraviolet CD spectrum of proteins can predict important characteristics of their secondary structure. CD spectra can be readily used to estimate the fraction of a molecule that is in the alpha-helix conformation, the beta-sheet conformation, the beta-turn conformation, or random conformation. These fractionalassignments place important constraints on the possible secondary conformations that the protein can be in.

Circular Dichroism UnitsThere are several different units of measurement for circular dichroism. Molar ellipticity, mean residue ellipticity and delta epsilons are all mentioned in the literature. Ellipticity is defined as the tangent of the ratio of the minor to major elliptical axes. More modern CD instruments measure the difference in absorption of right and left circularly polarized light as a function of wavelength. In accordance with the Beer–Lambert law, wavelength is equal to the difference in molar extinction coefficients divided by the product of path length and concentration. Mean residue ellipticity is the most common unit (degree cm2 dmol–1) and delta epsilons are the new machine unit, often referred to as molar circular dichroism (liter mol–1 cm–1), not to be confused with molar ellipticity(degrees decilitres mol–1 decimeter–1).

Application to Protein Secondary Structure

Precisely as done for infraredspectroscopy, we can correlatecertain UV-CD lineshapes withprotein structure. This permitsone to fit the CD spectrum ofa protein of unknown structureto a library of known structures.

Hence, UV-CD is a tool for the determination of secondarystructure in proteins.