Circular Dichroism - Indiana University

Circular Circular DichroismDichroism

What Can We Get from the CD What Can We Get from the CD Analysis of Proteins?Analysis of Proteins?

Part 1: What is CD?Part 1: What is CD?

Part 2: CD in analysis of Part 2: CD in analysis of protein folding, nonprotein folding, non--folding, folding, and and misfoldingmisfolding

OutlineOutline

Introduction to the Introduction to the circular circular dichroismdichroism

phenomenonphenomenon

What is CD?What is CD? Objectives #1:Objectives #1:

What is light? Electromagnetic waves What is light? Electromagnetic waves and types of polarizationand types of polarizationInteraction of light and matterInteraction of light and matterCircular Circular DichroismDichroism (CD) Spectroscopy(CD) SpectroscopyInstrumentationInstrumentationPhysics of CDPhysics of CDSample preparation and measurement Sample preparation and measurement

What is CD?What is CD? Objectives #2:Objectives #2:

CD of proteinsCD of proteinsFarFar--UV CD and secondary structureUV CD and secondary structureNearNear--UV CDUV CDContribution of aromatics in farContribution of aromatics in far--UV CDUV CDCD and conformational changesCD and conformational changes

Light as an electromagnetic waveLight as an electromagnetic waveTransmission of energy through a vacuum or using no medium is acTransmission of energy through a vacuum or using no medium is accomplished complished by by electromagnetic waveselectromagnetic waves, caused by the , caused by the osscilationosscilation of electric and magnetic of electric and magnetic fields. They move at a constant speed of 3x10fields. They move at a constant speed of 3x1088 m/sm/s. These electromagnetic . These electromagnetic waves are known as waves are known as electromagnetic radiation or light.electromagnetic radiation or light.

PlanePlane--Polarized Waves #1Polarized Waves #1

If the vector of the If the vector of the electric field oscillates electric field oscillates along a straight line along a straight line then the waves are then the waves are called called planeplane--polarizedpolarized or or linearly linearly polarizedpolarized waves. The waves. The following two slides following two slides represent animations represent animations showing a wave that is showing a wave that is planeplane--polarized in a polarized in a verticalvertical plane and in a plane and in a horizontalhorizontal plane. plane.


By By AndrAndrááss SzilSziláágyigyi



When two electromagnetic waves polarized in two When two electromagnetic waves polarized in two perpendicular planes are present simultaneously then the perpendicular planes are present simultaneously then the electric fields are added according to the rules of vector electric fields are added according to the rules of vector addition, 'parallelogram rule' (superposition). addition, 'parallelogram rule' (superposition).

The properties of the resulting electromagnetic wave depend The properties of the resulting electromagnetic wave depend on the intensities and phase difference of the component on the intensities and phase difference of the component waves. The following animations present the waves. The following animations present the superpositionssuperpositionsof two waves that have the same amplitude and wavelength, of two waves that have the same amplitude and wavelength, are polarized in two perpendicular planes and oscillate in are polarized in two perpendicular planes and oscillate in the the same phasesame phase or or have phase differencehave phase difference. Oscillating in the same . Oscillating in the same phase means that the two waves reach their peaks and cross phase means that the two waves reach their peaks and cross the zero line in the same moments. the zero line in the same moments.

Superposition of Two PlaneSuperposition of Two Plane--Polarized Waves #1Polarized Waves #1

Superposition of Two PlaneSuperposition of Two Plane--Polarized Polarized Waves #2Waves #2

The superposition of two waves that have the same amplitude and The superposition of two waves that have the same amplitude and wavelength, are polarized in two perpendicular planes and oscillwavelength, are polarized in two perpendicular planes and oscillate in ate in the same phasethe same phase


When two waves planeWhen two waves plane--polarized in two perpendicular planes polarized in two perpendicular planes meet out of phase then the wave resulting from the meet out of phase then the wave resulting from the superposition of the two waves will no longer be planesuperposition of the two waves will no longer be plane--polarized. polarized. The following animations present the superposition of two The following animations present the superposition of two waves that have the same amplitude and wavelength and are waves that have the same amplitude and wavelength and are polarized in two perpendicular planes but there is a phase polarized in two perpendicular planes but there is a phase difference of 90difference of 90°° and and --9090°° between them. A phase difference between them. A phase difference of 90of 90°° means that when one wave is at its peak then the means that when one wave is at its peak then the other one is just crossing the zero line. A phase difference of other one is just crossing the zero line. A phase difference of --9090°° requires shifting of one wave relative to the other one requires shifting of one wave relative to the other one along their axis so that there is a 3/4 wavelength difference along their axis so that there is a 3/4 wavelength difference between them. between them.

Superposition of Two PlaneSuperposition of Two Plane--Polarized Waves #3Polarized Waves #3


The superposition of two waves that have the same amplitude and The superposition of two waves that have the same amplitude and wavelength, are polarized in two perpendicular planes wavelength, are polarized in two perpendicular planes but there is a but there is a phase difference of 90 degrees between themphase difference of 90 degrees between them



The superposition of two waves that have the same amplitude and The superposition of two waves that have the same amplitude and wavelength, are polarized in two perpendicular planes wavelength, are polarized in two perpendicular planes but there is a but there is a phase difference of phase difference of --90 degrees between them90 degrees between them


Circularly Polarized WavesCircularly Polarized Waves By By AndrAndrááss SzilSziláágyigyi

Superposition of Circularly Superposition of Circularly Polarized WavesPolarized Waves

The superposition of a left circularly polarized wave and a right circularly polarized wave with equal amplitudes and wavelengths gives a plane-polarized wave.By thinking "backwards“: Any linearly polarized light wave can be obtained as a superposition of a left circularly polarized and a right circularly polarized light wave, whose amplitude is identical.


The Interaction of Light and MatterThe Interaction of Light and Matter

If light enters matter, its properties may change. Namely, If light enters matter, its properties may change. Namely, its intensity (amplitude), polarization, velocity, wavelength, its intensity (amplitude), polarization, velocity, wavelength, etc. may alter. The two basic phenomena of the interaction etc. may alter. The two basic phenomena of the interaction of light and matter are of light and matter are absorptionabsorption (or (or extinctionextinction) and a ) and a decrease in velocitydecrease in velocity. . AbsorptionAbsorption means that the intensity (amplitude) of light means that the intensity (amplitude) of light decreases in matter because matter absorbs a part of the decreases in matter because matter absorbs a part of the light. light. The decrease in velocityThe decrease in velocity (i.e. the slowdown) of light in (i.e. the slowdown) of light in matter is determined by a matter is determined by a refraction index.refraction index. The The refraction index is the ratio of the velocities of light refraction index is the ratio of the velocities of light measured in vacuum and in the given material.measured in vacuum and in the given material.

PlanePlane--Polarized Waves in an Absorbing Polarized Waves in an Absorbing MediumMedium


The following animation The following animation shows what happens shows what happens when a planewhen a plane--polarized polarized wave traverses a medium wave traverses a medium that absorbs light but that absorbs light but does not refract itdoes not refract it

Circularly Polarized Waves in an Circularly Polarized Waves in an Absorbing MediumAbsorbing Medium


This animation shows This animation shows what happens when a what happens when a circularly polarized circularly polarized light wave passes light wave passes throughout a lightthroughout a light--absorbing medium absorbing medium

PlanePlane--Polarized Waves in a Polarized Waves in a Refracting MediumRefracting Medium


The animation shows The animation shows what happens when a what happens when a planeplane--polarized wave polarized wave traverses a nontraverses a non--absorbing medium with absorbing medium with a refraction index a refraction index greater than 1.0.greater than 1.0.

Circularly Polarized Waves in a Circularly Polarized Waves in a Refracting MediumRefracting Medium By By AndrAndrááss SzilSziláágyigyi

The animation shows The animation shows what happens when a what happens when a circularly polarized circularly polarized wave traverses a nonwave traverses a non--absorbing medium with absorbing medium with a refraction index a refraction index greater than 1.0. greater than 1.0.

PlanePlane--Polarized Waves in a Medium Polarized Waves in a Medium Showing Circular Showing Circular DichroismDichroism #1#1

Some materials possess a special property: Some materials possess a special property: they absorb left they absorb left circularly polarized light to a different extent than right circularly polarized light to a different extent than right circularly polarized lightcircularly polarized light. This phenomenon is called . This phenomenon is called circular circular dichroismdichroism. .

As discussed, any linearly polarized light can be obtained as As discussed, any linearly polarized light can be obtained as the superposition of a left circularly polarized and a right the superposition of a left circularly polarized and a right circularly polarized light wave. Therefore, if linearly polarizecircularly polarized light wave. Therefore, if linearly polarized d light traverses a medium that shows circular light traverses a medium that shows circular dichroismdichroism, its , its properties will change because the medium absorbs the two properties will change because the medium absorbs the two circularly polarized components to a different extent. The circularly polarized components to a different extent. The next animation shows what happened.next animation shows what happened.

PlanePlane--Polarized Waves in a Medium Polarized Waves in a Medium Showing Circular Showing Circular DichroismDichroism #2#2


The left circularly The left circularly polarized component polarized component is not absorbed at is not absorbed at all (shown in red) all (shown in red) but the right but the right circularly polarized circularly polarized component is highly component is highly absorbed (shown in absorbed (shown in green) green)

The red component traverses the The red component traverses the medium unchanged and the green medium unchanged and the green component gets fainter. The component gets fainter. The superposition of the two components is superposition of the two components is no longer a linearly polarized wave: the no longer a linearly polarized wave: the resulting field vector does not oscillate resulting field vector does not oscillate along a straight line but it along a straight line but it rotates along rotates along and ellipsoid pathand ellipsoid path. Such a light wave is . Such a light wave is called an called an elliptically polarized lightelliptically polarized light. .

PlanePlane--Polarized Waves in a Medium Polarized Waves in a Medium Showing Circular Birefringence #1Showing Circular Birefringence #1

There are materials having another special property: There are materials having another special property: their their refraction index is different for left and right circularly refraction index is different for left and right circularly polarized lightpolarized light. This phenomenon is called . This phenomenon is called circular circular birefringencebirefringence. . As discussed earlier, linearly polarized light can be obtained aAs discussed earlier, linearly polarized light can be obtained as s the superposition of a left circularly polarized and a right the superposition of a left circularly polarized and a right circularly polarized light wave. Therefore, if linearly polarizecircularly polarized light wave. Therefore, if linearly polarized d light traverses a medium that shows circular birefringence, its light traverses a medium that shows circular birefringence, its properties will change because the two circularly polarized properties will change because the two circularly polarized components slow down in the medium to a different extent. components slow down in the medium to a different extent. Circular birefringence rotates the plane of polarization Circular birefringence rotates the plane of polarization of planeof plane--polarized lightpolarized light. .

PlanePlane--Polarized Waves in a Medium Polarized Waves in a Medium Showing Circular Birefringence #2Showing Circular Birefringence #2


The passing wave The passing wave continues to be a planecontinues to be a plane--polarized but its plane of polarized but its plane of polarization is no longer polarization is no longer vertical vertical –– it is it is rotatedrotated to to some anglesome angle

PlanePlane--Polarized Waves in aPolarized Waves in a Medium Medium Showing Both Circular Showing Both Circular DichroismDichroism and and Circular Birefringence #1Circular Birefringence #1

In reality, it rarely occurs that a material exhibits circular In reality, it rarely occurs that a material exhibits circular dichroismdichroism but no circular birefringence or it exhibits circular but no circular birefringence or it exhibits circular birefringence but no circular birefringence but no circular dichroismdichroism with respect to light of with respect to light of a certain wavelength. a certain wavelength.

The incident light suffers two modifications here: because of The incident light suffers two modifications here: because of the circular the circular dichroismdichroism, it becomes elliptical; and because of , it becomes elliptical; and because of the circular birefringence, its polarization gets rotated. Sincethe circular birefringence, its polarization gets rotated. Sincethe exiting light is no longer planethe exiting light is no longer plane--polarized, it is not the polarized, it is not the plane of polarization that gets rotated but the big axis of the plane of polarization that gets rotated but the big axis of the ellipse of polarization of the elliptically polarized light. ellipse of polarization of the elliptically polarized light.

PlanePlane--Polarized Waves in a Medium Polarized Waves in a Medium Showing Both Circular Showing Both Circular DichroismDichroism and and Circular Birefringence #2Circular Birefringence #2 By By AndrAndrááss SzilSziláágyigyi

Circular Circular dichroismdichroism and circular and circular birefringence are caused by the birefringence are caused by the asymmetry of the molecular asymmetry of the molecular structure of matter. The optical structure of matter. The optical activity of solutions of biological activity of solutions of biological macromolecules provides macromolecules provides information about the structural information about the structural properties of the macromolecules. properties of the macromolecules.

Circular Circular DichroismDichroism (CD) Spectroscopy(CD) Spectroscopy

CD is observed when optically active matter CD is observed when optically active matter absorbs left and right hand circular polarized light absorbs left and right hand circular polarized light slightly differently. slightly differently. The difference in left and right handed absorbance The difference in left and right handed absorbance AA(l)(l)-- AA(r) is very small (usually in the range of (r) is very small (usually in the range of 0.0001) corresponding to an 0.0001) corresponding to an ellipticityellipticity of a few of a few 1/100th of a degree. 1/100th of a degree. The CD is a function of wavelength. CD spectra for The CD is a function of wavelength. CD spectra for distinct types of secondary structure present in distinct types of secondary structure present in peptides and proteins are different. peptides and proteins are different. The analysis of CD spectra can therefore yield The analysis of CD spectra can therefore yield valuable information about secondary structure of valuable information about secondary structure of biological macromolecules. biological macromolecules.

Physics of CD and ORD #1Physics of CD and ORD #1

Physics of CD and ORD #2Physics of CD and ORD #2

In an optically active sample with a different In an optically active sample with a different absorbance absorbance AA for the for the two components, the amplitude of the stronger absorbed componenttwo components, the amplitude of the stronger absorbed component will will be smaller than that of the less absorbed component. be smaller than that of the less absorbed component.

A projection of the resulting amplitude yields an ellipse. Note A projection of the resulting amplitude yields an ellipse. Note that the that the polarization direction has not changed. The occurrence of polarization direction has not changed. The occurrence of ellipticityellipticity is is called called CircularCircular DichroismDichroism..

Rotation of the polarization plane (or the axes of the Rotation of the polarization plane (or the axes of the dichroicdichroic ellipse) ellipse) by a small angle by a small angle aa occurs when the phases for the 2 circular components occurs when the phases for the 2 circular components become different, which requires a difference in the refractive become different, which requires a difference in the refractive index index nn. . This effect is called This effect is called circular birefringencecircular birefringence. The change of optical . The change of optical rotation with wavelength is called rotation with wavelength is called optical rotaryoptical rotary dispersiondispersion. .

When CD exists, When CD exists, optical rotationoptical rotation must exist as well, and they are must exist as well, and they are directly related by a directly related by a KronigKronig--KramersKramers transformation. transformation.

CD Data Analysis CD Data Analysis

The difference in absorption to be measured is very small. The The difference in absorption to be measured is very small. The differential absorption is usually a few 1/100ths to a few 1/10tdifferential absorption is usually a few 1/100ths to a few 1/10th h of a percent, but it can be determined quite accurately. The rawof a percent, but it can be determined quite accurately. The rawdata represent the data represent the ellipticityellipticity of the sample in of the sample in millidegreesmillidegrees: :

To be able to compare these To be able to compare these ellipticityellipticity values they have to be values they have to be converted into a normalized value, the mean molar converted into a normalized value, the mean molar ellipticityellipticityper residue. We need to consider path length per residue. We need to consider path length ll, concentration , concentration cc , molecular mass , molecular mass MM and number of residues and number of residues nnrr::

InstrumentationInstrumentation

Instrumentation #2Instrumentation #2

Nitrogen purgingNitrogen purging

The function of purging the CD instrument with The function of purging the CD instrument with nitrogen is to remove oxygen from the lamp nitrogen is to remove oxygen from the lamp housing, housing, monochromatormonochromator, and the sample , and the sample chamber. chamber. The presence of oxygen is detrimental for two The presence of oxygen is detrimental for two reasons. When deep ultraviolet light strikes reasons. When deep ultraviolet light strikes oxygen, ozone is produced. Ozone causes oxygen, ozone is produced. Ozone causes degradation of optics and can cause respiratory degradation of optics and can cause respiratory problems. The second reason for removing oxygen problems. The second reason for removing oxygen is that oxygen absorbs deep UV light, thus is that oxygen absorbs deep UV light, thus reducing the light available for the measurement.reducing the light available for the measurement.

Sample preparation and measurementSample preparation and measurement

Additives, buffers and stabilizing compounds:Additives, buffers and stabilizing compounds: Any Any compound which absorbs in the region of interest (250 compound which absorbs in the region of interest (250 -- 190 190 nm) should be avoided (see below).nm) should be avoided (see below).

Protein solution:Protein solution: The protein solution should contain only The protein solution should contain only those chemicals necessary to maintain protein stability, and those chemicals necessary to maintain protein stability, and at the lowest concentrations possibleat the lowest concentrations possible.. AAny additional protein ny additional protein or peptide will contribute to the CD signal.or peptide will contribute to the CD signal.

Data collection:Data collection: Initial experiments are useful to establish Initial experiments are useful to establish the best conditions for the "real" experiment. Cells of 0.5 mm the best conditions for the "real" experiment. Cells of 0.5 mm path length offer a good starting point. path length offer a good starting point.

Typical Initial Conditions:Typical Initial Conditions:

Protein ConcentrationProtein Concentration: 0.5 mg/ml: 0.5 mg/mlCell Path LengthCell Path Length: 0.5 mm: 0.5 mmStabilizers (Metal ions, etc.):Stabilizers (Metal ions, etc.): minimumminimumBuffer Concentration :Buffer Concentration : 5 5 mMmM or as low or as low as possible while maintaining protein as possible while maintaining protein stabilitystability

Sample Concentration EffectsSample Concentration Effects

There is There is an optimum absorbancean optimum absorbance to use (Abs = 0.89). For to use (Abs = 0.89). For a 1 mm a 1 mm pathlengthpathlength cell, this absorbance is achieved with a cell, this absorbance is achieved with a protein concentration of about protein concentration of about 0.10.1--0.3 mg/ml0.3 mg/ml..

Cutoff Wavelengths For Common Cutoff Wavelengths For Common Solvents and BuffersSolvents and Buffers

WaterTrifluoroethanolHexafluoroisopropanolAcetonitrileMethanolEthanol2-PropanolCyclohexaneDimethylsulfoxideDioxane

<185<185<185185195196196

<185251232

(NH4)2SO4 0.15 MNaCl 0.15 MNaClO4 0.15 MNaNO3 0.15 M

191196

<185245

Phosphate 100 mMTris 100 mMPipes 100 mMMes 100 mM

<185195215205

GdnHCl 4 MUrea 4 M

210210

Any compound which absorbs in Any compound which absorbs in the region of interest (250 the region of interest (250 -- 190 190 nm) should be avoided. A buffer nm) should be avoided. A buffer or detergent or other chemical or detergent or other chemical should not be used unless it can should not be used unless it can be shown that the compound in be shown that the compound in question will not mask the protein question will not mask the protein signal. For instance signal. For instance imidazoleimidazolecannot be used below 220 nm cannot be used below 220 nm because it overwhelms the because it overwhelms the spectrum from then on. Therefore spectrum from then on. Therefore ensure that only the minimum ensure that only the minimum concentration of additives are concentration of additives are present in the protein solution.present in the protein solution.

(For one mm pathlength.)

CD of Proteins and PolypeptidesCD of Proteins and Polypeptides

For proteins we will be mainly dealing with the For proteins we will be mainly dealing with the absorption in the UV region of the spectrum absorption in the UV region of the spectrum originated from such originated from such chromophoreschromophores as peptide as peptide bond, amino acid side chains (aromatic side bond, amino acid side chains (aromatic side chains of chains of PhePhe, , TyrTyr, and , and TrpTrp have absorption have absorption bands in the vicinity of 250bands in the vicinity of 250--320 nm; the disulfide 320 nm; the disulfide group is an inherently asymmetric group is an inherently asymmetric chromophorechromophorethat can lead to a broad CD absorption around that can lead to a broad CD absorption around 250 nm), and any prosthetic groups.250 nm), and any prosthetic groups.

CD of Proteins. FarCD of Proteins. Far--UV Region #1UV Region #1

n n --> > ππ* centered around 220 nm* centered around 220 nm

ππ -->> ππ* centered around 190 nm* centered around 190 nm

n n -->> ππ* involves non* involves non--bonding bonding electrons of O of the carbonylelectrons of O of the carbonyl

ππ -->> ππ* involves the * involves the ππ--electrons of electrons of the carbonylthe carbonyl

The intensity and energy of The intensity and energy of these transitions depends on these transitions depends on φφand and ψψ (i.e., secondary structure)(i.e., secondary structure)



Far UVFar UV--CD of random coil:CD of random coil:

positive at 212 nm (positive at 212 nm (ππ-->>ππ*) *)

negative at 195 nm (nnegative at 195 nm (n-->>ππ*)*)

Far UVFar UV--CD of CD of ββ--sheet: sheet:

negative at 218 nm (negative at 218 nm (ππ-->>ππ*)*)

positive at 196 nm (npositive at 196 nm (n-->>ππ*)*)

Far UVFar UV--CD of CD of αα--helix:helix:

exitonexiton coupling of the coupling of the ππ-->>ππ* transitions leads * transitions leads to positive (to positive (ππ-->>ππ*)*)perpendicularperpendicular at 192 nm and at 192 nm and negative (negative (ππ-->>ππ*)*)parallelparallel at 209 nmat 209 nm

negative at 222 nm is red shifted (nnegative at 222 nm is red shifted (n-->>ππ*)*)

FarFar--UV CD Spectra and UV CD Spectra and Secondary StructureSecondary Structure

After baseline subtraction we are After baseline subtraction we are ready to analyze the data. Each ready to analyze the data. Each of the three basic secondary of the three basic secondary structures of a polypeptide chain structures of a polypeptide chain (helix, sheet, coil) show a (helix, sheet, coil) show a characteristic CD spectrum. A characteristic CD spectrum. A protein consisting of these protein consisting of these elements should therefore elements should therefore display a spectrum that can be display a spectrum that can be deconvoluteddeconvoluted into the individual into the individual contributions. contributions.

CD of Proteins. NearCD of Proteins. Near--UV RegionUV Region

If the aromatic residue is held rigidly in space If the aromatic residue is held rigidly in space than its environment is asymmetric, and it will than its environment is asymmetric, and it will exhibit circular exhibit circular dichroismdichroism. .

Aromatics have allowed Aromatics have allowed ππ-->>ππ* transitions (* transitions (11LLaaand and 11LLbb) that are directed in the plane of the ) that are directed in the plane of the ππ--bonding system and are orthogonal to each bonding system and are orthogonal to each other.other.

NearNear--UV CD: UV CD: PhenylalaninesPhenylalanines

PhePhe has a small extinction coefficient because of high has a small extinction coefficient because of high symmetry and it is also the least sensitive to alterations in symmetry and it is also the least sensitive to alterations in its environment. Absorption its environment. Absorption maximamaxima at 254, 256, 262 and at 254, 256, 262 and 267 nm (267 nm (vibronicvibronic bands).bands).

NearNear--UV CD: UV CD: TyrosinesTyrosines

TyrTyr has lower symmetry than has lower symmetry than PhePhe and therefore has more and therefore has more intense absorption band. intense absorption band. TyrTyr has absorption maximum at 276 has absorption maximum at 276 nm and a shoulder at 283 nm. Hydrogennm and a shoulder at 283 nm. Hydrogen--bonding to the bonding to the hydroxyl group leads to a redhydroxyl group leads to a red--shift of up to 4 nm. The dielectric shift of up to 4 nm. The dielectric constant may affect the spectrum too.constant may affect the spectrum too.

NearNear--UV CD: UV CD: TryptophansTryptophans

TrpTrp has the most intense absorption band centered at 282 has the most intense absorption band centered at 282 nm. Hydrogennm. Hydrogen--bonding to the NH can shift the bonding to the NH can shift the 11LLaa band by as band by as much as 12 nm.much as 12 nm.

NearNear--UV CD: SSUV CD: SS--BondsBonds

Disulfide (SDisulfide (S--S) spectra have a broad band at 250 S) spectra have a broad band at 250 -- 300 nm 300 nm with no with no vibronicvibronic structure.structure.

Contribution of Aromatics in FarContribution of Aromatics in Far--UV CDUV CDSpectra of Proteins #1Spectra of Proteins #1

PhePhe, , TyrTyr, , TrpTrp, and SS, and SS--bonds can complicate the bonds can complicate the farfar--UV CD region because UV CD region because of allowed of allowed ππ-->>ππ* * transitions, as illustrated bytransitions, as illustrated byvacuum ultraviolet CD vacuum ultraviolet CD spectra of the models of spectra of the models of aromatic side chain aromatic side chain residues. residues. GlutamylGlutamyl tyrosine tyrosine (Y); (Y); lysyllysyl--phenylalaninephenylalanine (F); (F); glutamylglutamyl--tryptophantryptophan (W).(W).

Contribution of Aromatics in FarContribution of Aromatics in Far--UV CDUV CDSpectra of Proteins #2Spectra of Proteins #2

Wavelength (nm)250 275 300 325

[ θ] (

deg

cm2 d

mol

-1)

-50

-25

0

25

50

75

100

HuIL-1β

Caf1

Wavelength (nm)200 210 220 230 240 250

[ θ]x

10-3

(deg

cm

2 dm

ol-1

)-4

-2

0

2

4

6

HuIL-1β

Caf1

CD and Conformational ChangesCD and Conformational Changes

Monitoring Monitoring θθ222nm222nm of a protein as a of a protein as a function of temperature or function of temperature or chemical denaturant yields chemical denaturant yields information about protein stability.information about protein stability.

The thermodynamic parameters, The thermodynamic parameters, ΔΔGGuu, , ΔΔHHuu, , ΔΔSSuu, T, Tmm, , ΔΔCCpp can be can be determined determined

CD and Conformational Changes: CD and Conformational Changes: Illustrative Examples #1Illustrative Examples #1

Wavelength (nm)250 275 300 325

[θ] (

ged

cm2 d

mol

-1)

-50

-25

0

25

50

75

100

Col 20 vs Col 21 Col 23 vs Col 24 Col 26 vs Col 27 Col 29 vs Col 30 Col 32 vs Col 33

pH

1 2 3 4 5 6 7 8

[ θ] λ

(deg

cm

2 dm

ol-1

)

0

20

40

60

80

100

[θ]268

[θ]273

[θ]287

[θ]294

pHpH--Induced Induced denaturationdenaturation of natively folded HuILof natively folded HuIL--11ββ

pHpH

CD and Conformational Changes: CD and Conformational Changes: Illustrative Examples #2Illustrative Examples #2

Wavelength (nm)

190 200 210 220 230 240 250

[θ] (

deg

cm2 d

mol

-1)

-30000

-20000

-10000

0

10000

[TFE] (%, v/v)0 10 20 30 40 50 60

[θ] 2

22 (d

eg c

m2

dmol

-1)

-20000

-15000

-10000

-5000

0

[θ] 1

98 (d

eg c

m2

dmol

-1)

-15000

-10000

-5000

0

TFETFE--induced folding of natively unfolded induced folding of natively unfolded αα--synucleinsynuclein

CD in analysis of protein CD in analysis of protein folding, nonfolding, non--folding and folding and

misfoldingmisfolding

Objectives #1:Objectives #1: A second part of A second part of the genetic codethe genetic code

Protein folding/Protein folding/misfoldingmisfolding/non/non--folding problem;folding problem;Mechanisms of protein folding;Mechanisms of protein folding;Partially folded intermediates Partially folded intermediates and protein folding/and protein folding/misfoldingmisfolding/ / nonnon--foldingfolding

Objectives #2: CD in protein folding/ Objectives #2: CD in protein folding/ misfoldingmisfolding/non/non--folding studiesfolding studies

ChickenChicken--egg scenario of protein egg scenario of protein foldingfoldingCD of molten globulesCD of molten globulesDiscriminating native coils and Discriminating native coils and native prenative pre--molten globules by CDmolten globules by CDCD of CD of amyloidogenicamyloidogenic intermediateintermediate

The route from the DNA code to The route from the DNA code to the protein the protein

The DNA is responsible for The DNA is responsible for coding for all proteins.coding for all proteins. Each Each amino acid is designated by amino acid is designated by one or more set of triplet one or more set of triplet nucleotides. The code is nucleotides. The code is produced from one strand of the produced from one strand of the DNA by a process called DNA by a process called "transcription". This produces "transcription". This produces mRNA which then is sent out of mRNA which then is sent out of the nucleus where the message the nucleus where the message is translated into proteins.is translated into proteins. The The cartoon to the left shows the cartoon to the left shows the basic sequence of transcription basic sequence of transcription and translational events. and translational events.

Fate of a polypeptide chainFate of a polypeptide chainUversky (2003) Uversky (2003) Cell. Mol. Life Cell. Mol. Life SciSci.. 60 185260 1852

PolypeptideChain Non-folding

Misfolding

Folding

Mystery of Protein Folding: Why?Mystery of Protein Folding: Why?

Side view of the Side view of the hexadecamerichexadecamericthermosomethermosome complexcomplex

Protein folds to Protein folds to be functionalbe functional

Mystery of Protein Folding: How?Mystery of Protein Folding: How?

Designed by T.A. Designed by T.A. BramleyBramley, , in in Robson, B., Robson, B., Trends Trends BiochemBiochem. . SciSci. . 1,50 (1976).1,50 (1976).Copyright (c) Elsevier Biomedical Press, 1976.Copyright (c) Elsevier Biomedical Press, 1976.

Protein folding through funnelsProtein folding through funnels

Folding funnel model:Folding funnel model:Each conformation is Each conformation is represented as a point represented as a point on the landscape. on the landscape. Folding is limited by a Folding is limited by a finding of an optimal finding of an optimal pathway throughout the pathway throughout the rugged landscaperugged landscape

Framework model of protein Framework model of protein foldingfolding

Unfolded First foldingUnfolded First folding Second folding NativeSecond folding Nativestatestate intermediateintermediate intermediateintermediate statestate

(Pre(Pre--molten molten (Molten globule)(Molten globule)globule)globule)

O.B. O.B. PtitsynPtitsyn DAN SSSRDAN SSSR 210 (1973) 1213210 (1973) 1213--12151215

The chickenThe chicken--egg scenario of egg scenario of protein folding revisited protein folding revisited

[θ]222/[θ]222U

0 2 4 6 8 10 12 14 16

(RSU

/RS )

3

0

2

4

6

8

10

12

14

16

Uversky & Fink (2002) Uversky & Fink (2002) FEBS FEBS LettLett.. 515, 79515, 79

No compact equilibrium intermediates lacking secondary structureNo compact equilibrium intermediates lacking secondary structure, or highly , or highly ordered nonordered non--compact species, were found. This means that the hydrophobic compact species, were found. This means that the hydrophobic collapse occurs simultaneously with formation of secondary struccollapse occurs simultaneously with formation of secondary structure in the ture in the early stages of the protein folding.early stages of the protein folding.

NativeNative--like secondary structure of like secondary structure of molten globulesmolten globules

Wavelength (nm)190 200 210 220 230 240

[ θ]x

10-3

deg

cm

2 dm

ol-1

-20

-10

0

10

20

30

40

NativeNative proteinsproteins

Wavelength (nm)190 200 210 220 230 240

[ θ]x

10-3

deg

cm

2 dm

ol-1

-20

-10

0

10

20

30

40

Molten globulesMolten globules

CD spectra analysis CD spectra analysis revealed the existence revealed the existence of a clear correlation of a clear correlation between the shape of between the shape of the molten globule farthe molten globule far--UV CD spectra and the UV CD spectra and the content of secondary content of secondary structure elements in structure elements in the corresponding the corresponding native proteins, as native proteins, as determined from Xdetermined from X--ray ray data.data. Thus, the Thus, the secondary structure of secondary structure of protein in the MG state protein in the MG state is close to that in the is close to that in the native state.native state.

Spectra need at least four independent components to be described

Spectra can be described as a superposition of three independent components (most likely α-, β- and irregular structure)

Vassilenko & Uversky (2002) BBA 1594, 168

Protein nonProtein non--folding problemfolding problem

Protein stays substantially unfoldedProtein stays substantially unfolded

Protein partially folds to a preProtein partially folds to a pre--molten molten globuleglobule--like statelike state

Protein partially folds to a molten Protein partially folds to a molten globuleglobule--like statelike state

Different levels of disorder in a Different levels of disorder in a protein moleculeprotein molecule

Different levels of order and disorder: (0) Different levels of order and disorder: (0) no disorder; (1) disordered Nno disorder; (1) disordered N-- and Cand C--termini; (2) disordered loop; (3) termini; (2) disordered loop; (3) disordered linker; (4) disordered domain; disordered linker; (4) disordered domain; (5) disordered protein with some residual (5) disordered protein with some residual structure; and (6) wholly disordered, structure; and (6) wholly disordered, mostly collapsed protein; and (7) wholly mostly collapsed protein; and (7) wholly disordered, mostly extended protein. disordered, mostly extended protein. Corresponding disordered regions are Corresponding disordered regions are shown in red.shown in red.

Uversky et al. (2005) J. Mol. Recognit. In pressUversky et al. (2005) Progress Biophys. Mol.

Biol. In press

Protein nonProtein non--folding: Why?folding: Why?

Mean hydrophobicity0.1 0.2 0.3 0.4 0.5 0.6

Mea

n ne

t cha

rge

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Natively folded

proteins

Natively unfoldedproteins

Natively unfolded Natively unfolded proteins possess proteins possess unique combination unique combination of low mean of low mean hydrophobicityhydrophobicityand high mean net and high mean net charge. High net charge. High net charge leads to charge leads to strong electrostatic strong electrostatic repulsion, and low repulsion, and low hydrophobicityhydrophobicity means means less driving force for less driving force for compaction.compaction.

Uversky et al. (2000) Proteins 42, 415

ChargeCharge--hydropathyhydropathy plotplot

Amino acid determinants of Amino acid determinants of protein protein foldabilityfoldability

-1

-0.5

0

0.5

1

W C F I Y V L H M A T R G Q S N P D E K

(Dis

orde

r –O

rder

) / O

rder

Dunker et al. (2002) Adv. Protein Chem. 62, 25

Order promoting residuesOrder promoting residues

dis XRAY (2844)dis XRAY (2844)dis NMR (4019)dis NMR (4019)dis CD (10554dis CD (10554))

Disorder promoting residuesDisorder promoting residues

When nonWhen non--folding meets foldingfolding meets folding

Folding induced by Folding induced by cationcation binding: binding: Experimental evidencesExperimental evidences

Wavelength (nm)

190 200 210 220 230 240 250

[θ]x

10-3

(deg

cm

2 dm

ol-1

)

-20

-10

0

10

20Core histones

NaCl

Wavelength (nm)

190 200 210 220 230 240 250

[ θ] (

deg

cm2 d

mol

-1)

-12000

-10000

-8000

-6000

-4000

-2000

0

2000

1 -No cation2 - 5.0 mM MnCl23 - 5.0 mM CdCl24 - 5.0 mM FeCl35 - 5.0 mM CoCl26 - 10 mM NaCl

α-Synuclein

1

2

3 4, 5

1, 6

Wavelength (nm)

190 200 210 220 230 240 250

[θ]x

10-3

(deg

cm

2 dm

ol-1

)

-25

-20

-15

-10

-5

0

1 - 0.00 mM ZnCl2

2 - 0.74 mM ZnCl2

3 - 1.55 mM ZnCl2

4 - 2.34 mM ZnCl2

5 - 3.13 mM ZnCl2

6 - 3.76 mM ZnCl2

7 - 4.74 mM ZnCl2

8 - 6.90 mM ZnCl2

9 - 9.40 mM ZnCl2

10 - 12.50 mM ZnCl2

Prothymosin αUversky et al. (2000) BBRC 267, 663Uversky et al. (2001) JBC 276, 44284Uversky et al. (2002) JPR 1, 149Permyakov et al. (2003) Proteins 53, 855Munishkina et al. (2004) JBC 342, 1305

Structural classification of natively Structural classification of natively unfolded proteinsunfolded proteins

log M (Da)3 5 7

log ρ

(Da/

Å3 )

-3

-2

-1

0

NMG

PMG

UGdmCl

NUcoil

Uurea

NUPMG

Uversky V.N. (2002) Uversky V.N. (2002) Protein Protein SciSci.. 11, 73911, 739Uversky V.N. (2002) Uversky V.N. (2002) Eur. J. Eur. J. BiochemBiochem. . 269, 2269, 2Uversky V.N. (2003) Uversky V.N. (2003) Cell. Mol. Life Cell. Mol. Life SciSci.. 60, 185260, 1852

[θ]200 (deg cm2 dmol-1)

-22500 -20000 -17500 -15000 -12500 -10000 -7500

[ θ] 22

2 (de

g cm

2 dm

ol-1

)

-8000

-7000

-6000

-5000

-4000

-3000

-2000

-1000

0

PMG-like

Coil-like

Protein misfolding and Protein misfolding and conformational disordersconformational disorders

Widespread phenomenon: more than Widespread phenomenon: more than 450 different disorders, more than 25 450 different disorders, more than 25 different proteins;different proteins;Prior to fibrillation, Prior to fibrillation, amyloidogenicamyloidogenicproteins have different structures proteins have different structures –– they they may be rich in may be rich in ββ--sheet, sheet, αα--helix, helix, ββ--helix, helix, or contain both or contain both αα--helices and helices and ββ--sheetssheetsThey may be globular proteins with rigid They may be globular proteins with rigid 3D3D--structure or be natively unfoldedstructure or be natively unfolded

Protein Protein misfoldingmisfolding problem problem

Ordered

Native MG

Native coilNative PMG Amyloid fibril

Protein misfolding: Protein misfolding: amyloidamyloidfibrilsfibrils

AmyloidosisAmyloidosis starts from proteins with different starts from proteins with different structures, but ends with very uniform structures, but ends with very uniform deposits. Why and how it happens?deposits. Why and how it happens?

Conformational prerequisites Conformational prerequisites for for amyloidosisamyloidosis #1#1

Uversky & Fink (2004) BBA 1698, 131

W avelength (nm)

190 200 210 220 230 240

[θ] (

deg

cm2 d

mol

-1)

-20000

-10000

0

10000

20000

30000

40000

[θ] (

deg

cm2 d

mol

-1)

-20000

-10000

0

10000

20000

30000

40000

W avelength (nm)

190 200 210 220 230 240

[θ] (

deg

cm2 d

mol

-1)

-20000

-10000

0

10000

20000

30000

40000

[θ] (

deg

cm2 d

mol

-1)

-20000

-10000

0

10000

20000

30000

40000A

B

C

D

Representative farRepresentative far--UV CD spectra of UV CD spectra of proteins in their native proteins in their native ((AA), molten globule ), molten globule ((BB), pre), pre--molten molten globule (globule (CC) and ) and spectra of proteins in spectra of proteins in their their amylodogenicamylodogenicconformation (conformation (DD).).

Note close similarity Note close similarity of of CC and and DD..

Conformational prerequisites Conformational prerequisites for for amyloidosisamyloidosis #2#2

[θ]200 (deg cm2 dmol-1)

-30000 -25000 -20000 -15000 -10000 -5000 0 5000 10000 15000 20000

[θ] 22

2 (de

g cm

2 dm

ol-1

)

-25000

-20000

-15000

-10000

-5000

0U

PMG

MG

N

AmyloidogenicAmyloidogenicintermediates can be intermediates can be distinguished by their distinguished by their secondary structure secondary structure properties. The figure properties. The figure compares the farcompares the far--UV CD UV CD spectral parameters spectral parameters measured for the measured for the amyloidogenicamyloidogenicconformations of 11 conformations of 11 proteins (red circles) to proteins (red circles) to those of proteins in other those of proteins in other defined conformations.defined conformations.

Uversky & Fink (2004) BBA 1698, 131

Fate of the polypeptide chainFate of the polypeptide chain

FoldingNon-folding

Misfolding

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