Welcome to Welcome to 2011 UK CD and LD winter 2011 UK CD and LD winter

workshopworkshop

Thanks to MOAC, Department of Thanks to MOAC, Department of Chemistry, and the University of Chemistry, and the University of

Warwick for hosting us.Warwick for hosting us.

SafetySafetyIn the lab: lab coats, safety In the lab: lab coats, safety

spectacles, gloves as appropriate.spectacles, gloves as appropriate.

In the event of MOAC fire alarm go In the event of MOAC fire alarm go outside the front door.outside the front door.

In event of chemistry fire alarm – we In event of chemistry fire alarm – we meet outside library. No lifts! meet outside library. No lifts!

Elsewhere read signs.Elsewhere read signs.

Where will we be??Where will we be??Lectures and computer workshopsLectures and computer workshops: : MOAC EPSRC Doctoral Training CentreMOAC EPSRC Doctoral Training Centre

Laboratory work:Laboratory work: Mainly chemistryMainly chemistry

FoodFood: : Lunches: DIY Lunches: DIY –– including clear-up! Use the time including clear-up! Use the time

to get to know other people.to get to know other people.Coffee/Tea/Squash: available Coffee/Tea/Squash: available –– again DIY. again DIY.Dishwasher need stacking with dirty dishes and Dishwasher need stacking with dirty dishes and

emptying when clean!emptying when clean!Tuesday dinner with, posters Tuesday dinner with, posters etcetc. in MOAC.. in MOAC.Wednesday: Browns in centre of the cityWednesday: Browns in centre of the city

More thingsMore things

Keep the common room tidyKeep the common room tidy

READ but not REMOVE books!READ but not REMOVE books!

Internet access via the common room Internet access via the common room computers. Ask about wireless if you computers. Ask about wireless if you have a laptop. have a laptop.

Wear your name badges.Wear your name badges.

Receipts/refunds on deposit see me of Receipts/refunds on deposit see me of you haven’t got one.you haven’t got one.

PeoplePeopleAll organisational credit to:All organisational credit to:

Anne Maynard and Fiona FrielAnne Maynard and Fiona Friel

Big problems: Alison Rodger Big problems: Alison Rodger

Warwick studentsWarwick students

Lecturers and course leaders – see programme.Lecturers and course leaders – see programme.

Alison Rodger, University of Alison Rodger, University of WarwickWarwick

a.rodger@warwick.ac.uk

Circular Dichroism Circular Dichroism Spectroscopy Spectroscopy Introduction Introduction

AbsorbanceAbsorbanceBonds Molecules SpectroscopyMolecules are 'glued together' by electrons between the atoms. ~ Two electrons per bond.

Two types of bonds are important for bio molecules: (sigma) bonds look like s-orbitals

when viewed along the bond axis. (pi) bonds look like p-orbitals (dumbbells)

when viewed down the bond axisAlso non-bonding pairs, e.g. lone pairs on N &

O

Ultra violet/visible (UV/vis): how much energy is required to push electrons to new orbitals.

Electrons Electrons bonds bonds structurestructure

.

UV/visible light: ~ 180 nm – 800 nm, energy hhc/causes electrons to go to higher energy levels.

required depends on electron rearrangement needed.

In solution: broad bands due to diff. vibrational levels in excited state &molecules having slightly different energy levels.

UV: –350 nmVis: 400 nm –

Excited electronic state

Ground electronic state

Ground vibn’l levelr

E

Absorption spectra of DNA Absorption spectra of DNA & proteins& proteinsWith proteins and DNA the transitions we usually With proteins and DNA the transitions we usually

study study are n are n * and * and * so can access them with * so can access them with normal spectrometers.normal spectrometers.

Below 200 nm need nitrogen purging because OBelow 200 nm need nitrogen purging because O22 absorbsabsorbs

Absorbance: A = log(Io/I) Absorbance: A = log(Io/I) = log(intensities in/out) = log(intensities in/out)

Beer Lambert A = Beer Lambert A = cl, cl, extinction extinction coefficient (units?),coefficient (units?),

c concentration, l length (cm)c concentration, l length (cm)

Linearly polarized lightLinearly polarized light

• All photons in a beam of light have an electric field vector, E, that is at right angles to the direction of travel of the beam, x, and varies as a sine wave

E

x

E

Before polarization:unpolarized beam po

lari

zer

After polarization:linear polarized beam

EE

Circularly polarized lightCircularly polarized light • Add two linearly

polarized light beams - both propagating along x

• y-polarized + • z-polarised but starting

¼ wavelength out of phase from the y one.

Ey= msin(2y/)

Ez= mcos(2z/)

z

y

z

y

xor time

The two waves add together to form right handed (clockwise) circularly polarized light.

Circular DichroismCircular Dichroism• CD is the difference between the absorption of left

and right handed circularly polarized light as a function of wavelength.

• The difference very small (~<<1/1000 of total) A() = AL()-AR() = [L() - R()]lc or

A() = ()lc

• ~ typically < 10 M-1cm-1 vs. ~20,000 M-1cm-1

CD is very small difference between two large signals

ACD

Circularly polarized lightCircularly polarized light

Linearly polarized light: Linearly polarized light:

Electric vector direction constant—magnitude varies.Electric vector direction constant—magnitude varies.

Circular polarized light:Circular polarized light:

Electric vector direction varies—magnitude constantElectric vector direction varies—magnitude constant

Varying absorption of circularly polarised light by chiral molecules results in distinctive spectra under

absorption bands

Need chiral light and chiral molecule to get CD spectrum of a solution

CD SpectraCD Spectra

CD spectropolarimeterCD spectropolarimeter

Quartz 1/4 -plate. Oscillatesat ~ 50 Hz CPL

Max light intensity: 300-400 nm

M = mirrorP=prism

Monochromaterprism

CD=A/(absorbance units)=4(degrees)/(180ln10)CD=(millidegrees)/32,980

Xenon lamp

Empirical analysis of CDEmpirical analysis of CD

HPLC detector

Structural change

CD to answer: does it CD to answer: does it change structure?change structure?

-20

-15

-10

-5

0

5

10

190 195 200 205 210 215 220 225 230 235 240

Wavelength (nm)

(md

eg

)

-20

-15

-10

-5

0

5

10

190 195 200 205 210 215 220 225 230 235 240

Wavelength (nm)

(md

eg

)

C ircu lar d ichro ism spectra of the p lant defensin H s-AFP 1 in w a ter/acetonitrile (1 :1, ) + 1% form ic acidv/v

U nreduced P lan t de fens in

C hem ica lly reduced p lant de fensin

Reduced and unreduced plant defensins

Two regionsTwo regions

Secondary structure (170 – 260 nm)Secondary structure (170 – 260 nm)

Aromatic region (240 – 360 nm)Aromatic region (240 – 360 nm)

The CD signal for a protein The CD signal for a protein depends on its secondary depends on its secondary structurestructure

180 200 220 240 260Wavelength/nm

-helix

-sheet

turn

Poly proline type II

20

15

10

5

0

5

10

Secondary structure- Amide backbone

Aromatic - Phenylalanine (250 – 270 nm) -Tryptophan (260 – 300 nm) - Tyrosine (270 – 290 nm) - Disulfide bonds -helical protein spectra are distinctive: 222,208,~190 nm

Amino acids, Amino acids, peptides peptides and proteinsand proteins

H

H

CD CD secondary structure of secondary structure of proteinprotein

• Fit the unknown CD curve u to

a combination of standard curves

• In the simplest case use the standard spectra for secondary structures

t = x + x + xcc

• Vary xx and xc to give the

best fit of t to u

• while x+ x + xc = 1.0 • fits best with: x= 80%x=0%; xc=

20%

• agrees well with structure:

78% helix, 22% other

CD spectra can be analysed by the structure-fitting program “cdsstr” (of C.J. Johnson) to obtain % of secondary structure motifs.

Cdsstr uses a basis set of protein spectra

CD signals are sensitive to CD signals are sensitive to secondary structure: coiled-coil (2 secondary structure: coiled-coil (2 --helices twisted)helices twisted)

Characteristic heptad repeat Characteristic heptad repeat

aabcbcddefgefgaabcbcddefgefgaabcbcddefgefgaabcbcddefgefgMMKQKQLLEDKEDKVVEEEELLLSKNYHLSKNYHLLENEENEVVARARLLKKLKKL

hydrophobic

Salt bridge

The CD signal for a protein depends on The CD signal for a protein depends on its secondary structureits secondary structure

—— chymotrypsin (~0.1 helix, 0.15 sheet0.15turn)—— lysozyme (mixed 0.4 helix, 0.2 turn)—— triosephosphate isomerase (mostly some )—— myoglobin (all )

Protein conformation as a Protein conformation as a function of environmentfunction of environment

-3

-2

-1

0

1

2

3

4

5

190 200 210 220 230 240 250 260

Wavelength / nm

Mol

ar R

esid

ue e

llipt

icity

Tris buffer soluble Pre-PSbW and and Unfolded using Guanidinium Chloride

-4

-3

-2

-1

0

1

2

3

4

5

190 200 210 220 230 240 250 260

Wavelength / nm

SPP TPP Transmembrane -helix

N C

Hydrophobic region

Pre-PsbW, thylakoid membrane protein

‘No structure’ in guanidinium chlorideSome in tris buffer (multimer)Lots in SDS micelles (folded, 2 helices)

CDsstr results for pre-CDsstr results for pre-PsbWPsbW

0

10

20

30

40

50

60

Number of residues

Pre-PSbW(SDS)

Pre-PSbW (OG) PSbW (OG)

Helical

Strand

Turn

Other

- - -

Lots of Lots of -helix-helix

1.4 mg/mL; 0.01 cm;42% α-helix208 nm, 100% -helix = 12 -helix = 12 molmol-1-1dmdm33cmcm-1-1

Averaged CD spectrum of the sample 67544

-30

-20

-10

0

10

20

30

40

190 210 230 250 270 290 310

Wavelenghth/nm

CD

/mde

g

AntibodyAntibody

0% α-helix10% other helix33% β-sheet14% turn42% other

Largely Largely -sheet protein-sheet proteindE spectra of samles 71603-71638 using a 0.1mm cuvette

-5

-4

-3

-2

-1

0

1

2

190

194

198

202

206

210

214

218

222

226

230

234

238

242

246

250

254

258

Wavelength (nm)

dE

(m

ol-1

dm

3 cm

-1)

0.01 cm; 0.3 mg/mL;16% PPII19% β-sheet15% turns47% other

Typical mixed Typical mixed -sheet protein -sheet protein spectrumspectrum

dE - Sample 71004 subtracting the pH 7.2 buffer baseline

-3-2-1012345

190

194

198

202

206

210

214

218

222

226

230

234

238

242

246

250

254

258

Wavelength (nm)

dE

mo

l-1 d

m3 c

m-1

15% α-helix11% other helix26% β-sheet12% turns36% other

Near UV: protein CDNear UV: protein CD

CD requires helical CD requires helical electron motion electron motion

Require magnetic dipole transition moment 0Require electric dipole transition moment 0

Im 0 0R f f μ m

CD from coupled CD from coupled oscillatorsoscillators

R = CD strength = Im(.m)=electric dipole transition momentm=magnetic dipole transition moment

High energy, 190 nmLow energy, 208 nm

-* transitionof a helical polypeptide

Oriented CD spectraOriented CD spectra

Vancomycin & ristocetinVancomycin & ristocetinGlycopeptide antibiotics that prevent cross-linking and

transglycosylation during bacterial cell wall formation.Noncovalent dimerisation plays a key role in their activityCD used to give binding constants V-V, V-R and

V-peptides, R-peptides. Assume non-covalent dimers.

Circular dichroism spectra for the Titration of Vancomycin with Ristocetin

-60

-50

-40

-30

-20

-10

0

10

20

30

40

50

190 210 230 250 270 290 310

Wavelength (nm)

Inte

ns

ity

(m

de

g)

[25uM vanco + 25uM risto]

[50uM vanco + 25uM risto]

[75uM vanco + 25uM risto]

[100uM vanco + 25uM risto]

[125uM vanco + 25uM risto]

[150uM vanco + 25uM risto]

[175uM vanco + 25uM risto]

[200uM vanco + 25uM risto]

Circular dichroism spectra for the Titration of Vancomycin with Ristocetin

-60

-50

-40

-30

-20

-10

0

10

20

30

40

50

190 210 230 250 270 290 310

Wavelength (nm)

Inte

ns

ity

(m

de

g)

[25uM vanco + 25uM risto]

[50uM vanco + 25uM risto]

[75uM vanco + 25uM risto]

[100uM vanco + 25uM risto]

[125uM vanco + 25uM risto]

[150uM vanco + 25uM risto]

[175uM vanco + 25uM risto]

[200uM vanco + 25uM risto]

CD change (induced CD) [dimer]

Kdimerisation= 205 (mM)-1

V + R V-R

Nucleic acid CDNucleic acid CDDNA and RNA polymers: sugar units of the backbone

provide the chirality, but not the chromophores.CD spectrum of a polynucleotide arises from interaction

between the * transitions of stacked bases.But note isolated nucleotides are chiral

Use CD to identify which polymorph: CD varies more with base orientation than sequence

Nucleic acid CD spectraNucleic acid CD spectra

B-DNA: 72%, 50% & 31% GC content

Calf thymus DNA: B-DNA (10.4 bases) A-DNA B-DNA (10.2 bases)

Poly[d(G-C)] 2:

B-DNA A-DNA Z-DNA

B-DNA: 275>0, 258=0, 240<0, 220>/=0, 180/190>>>0A:DNA: 295</=0, 260>>0, 250230>/=0, 210<<0, 190>>0Z-DNA: 290<0, 260>0, 195/200<<<0, 185 180=0

RNA: CNG repeats RNA: CNG repeats (neurological disorders e.g. (neurological disorders e.g. Myotonic DystrophyMyotonic Dystrophy ) )

Unusual RNAs: adopt duplex A-form plus something else. ??? Triplex.

Which is melted?

5'

3'

[Ru(phen)[Ru(phen)33]]2+2+ct+high r

ct+low r

AT

GC