Circular Dichroism Spectroscopy with Synchrotron Radiation

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Circular Dichroism Spectroscopy with Synchrotron
Radiation
September 2006
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Optical activity

• Ability of a substance to rotate the plane of
  polarization of a beam of light passed through
  it.

Jean-Baptiste Biot (1774-1862) suggested optical
rotation arises from asymmetric or chiral
molecules. Later verified by Pasteur using
tartrate crystals.
Biots original polariscope
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Polarization
The property of electromagnetic waves that
describes the direction of their transverse
electric field. More generally, the
polarization of a transverse wave describes the
direction of oscillation in the plane
perpendicular to the direction of travel.
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Optical activity in biology
Proteins are made up of chains of amino acids.
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Levels of protein structure
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Protein secondary structure
Local structure of linear segments of the
polypeptide backbone atoms.
Alpha helix
310 helix
Beta strand
Type I Beta turn
Type II Beta turn
Phi -57.8 -74.0 -139.0
-60 (i1) -90 (i2)
-60 (i1) 80 (i2) Psi -47.0 -4.0
135.0 -30
(i1) 0 (i2) 120 (i1) 0 (i2)
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Optical activity in biology
In proteins, optical activity arises from the
asymmetric a-carbon atoms of the peptide bond.
This chromophore absorbs in the
vacuum-ultraviolet and ultraviolet region of the
spectrum (140-260 nm).
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Circular Dichroism
Circular dichroism (CD) is the difference in
absorption between left and right circularly
polarised light by a chiral chromophore.
?e el - er Optical rotation
(ORD) and CD contain the same information and are
related by Kronig-Kramers Transforms. CD data
are more easily interpreted than optical rotation
data because it is an absorption rather than
dispersion phenomenon.
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Circular Polarization
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Circular Dichroism
In proteins, CD of the amide chromophore reports
on secondary structure (proportions of alpha
helix, beta sheet, turns, etc.).
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Secondary Structure from CD
The CD spectrum of a protein, S(?), can be
analysed as a linear combination of k basis
spectra, Bk(?) (for 100 helix, etc.)
Where N is the number of secondary structures and
fk the fraction of the kth secondary
structure. The basis spectra Bk(?) are
calculated from CD spectra of a set of reference
proteins of known three-dimensional structure. A
number of methods have been developed to
determine the best fit of the basis spectra to
the spectrum being analysed.
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Applications of CD in structural biology

• Determination of secondary structure of proteins
  that cannot be crystallised
• Investigation of the effect of e.g. drug binding
  on protein secondary structure
• Dynamic processes, e.g. protein folding
• Studies of the effects of crystallisation media
  on protein structure
• Secondary structure of membrane proteins
• Study of active site changes
• Carbohydrate structure

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How is CD measured?
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The CD Signal
For biological samples, IAC is typically 104
times smaller than IDC.
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CD Instrumentation
Cary 60 CD machine (circa 1965) Modulation by
Pockels cell
Spectrum of BSA run on Cary 60
Jasco J-810 (2003) Modulation by photoelastic mod
ulator (PEM)
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How could we do better?
We need a lamp that has High brightness Extended
wavelength range (VUV-Near IR) Inherent
polarization
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Synchrotron radiation
SRCD spectrum of DNA/benzo(a)pyrene adduct
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CD12
First 2nd Generation SRCD facility.
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What can SRCD do?
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Implications of SRCD data
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Leucine-Rich Repeat Peptides
Leucine-rich repeat (LRR) pXXXFXXLXXLXXLXLXXNXIX
XL Between 1 and 38 tandemly repeated
copies. e.g. Ribonuclease Inhibitor
LRRN Peptide PANLLTDMRNLSHELRANIEEM Forms
spontaneously into gels with ß-sheet
structure. X-ray fibre diffraction shows a
cross-ß structure, similar to Alzheimer
ß-amyloid and prion protein fibrils.
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Leucine-Rich Repeat Peptides
SRCD spectra recorded during LRRN
polymerisation. Spectra recorded at 20 minute
intervals.
De (M-1 cm-1 per residue)
Wavelength (nm)
This residue is fundamental to the polymerisation
process.
PANLLTDMRNLSHELRANIEEM
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SRCD for drug discovery
Captopril and Lisinopril are two commonly used
drugs for the treatment of hypertension. They
bind to angiotensin converting enzyme (ACE),
inhibiting the activation of the vasoconstrictor,
angiotensin.
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SRCD for drug discovery
Analysis shows major secondary structure
components in agreement with x-ray. No
significant secondary structure changes on drug
binding.
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SRCD for drug discovery
The left-handed polyproline II helix has a CD
minimum around 198 nm.
The most likely explanation for changes in the CD
spectrum of ACE with drugs is the adoption of a
left-handed helix by the drug molecule. The
effect is more pronounced with the 3-residue
analogue lisinopril. G.R. Jones and D.T. Clarke,
Faraday Discuss., 2003, in press.
Lisinopril forms an extended left-handed helix
when complexed with ACE. R. Natesh et al.,
Nature 421, 551 (2003).
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Time-resolved CD
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The Protein Folding Problem
?
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Time-resolved CD Protein folding
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Protein folding energy landscape
Radford, TIBS 25, 581, 2000.
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Initiation of fast processes
DL Stopped-Flow Apparatus Dead time 600
microseconds Mixing ratios 11 to 201 Sample
volume per shot 20-80 microlitres
(depending on mixing ratio) Operating
pressure 0.5 bar in the cell Drive system
compressed gas Minimum pathlength 0.5 mm
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Beta-lactoglobulin
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Beta-lactoglobulin secondary structure prediction
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Time-resolved SRCD
Folding of b-lactoglobulin
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Time-resolved CD
Folding of b-lactoglobulin secondary structure
development
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Laser temperature-jump
NdYAG laser. 1064 nm fundamental Raman shifted
to 1543 nm. 100 mJ in a 10 ns pulse. Heating
around 20C for 20µl.
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Myoglobin folding T-jump and stopped-flow
(Temperature jump -7.2 to 10 C)
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Flash Photolysis
NdYAG laser. 1064 nm fundamental 2x frequency
doubled to 266 nm. 10 mJ in a 10 ns pulse. 532
nm harmonic could also be Made available.
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Flash Photolysis
Waltho group, Sheffield. Photolysis system based
on a bifunctional aromatic disulphide compound
that cross-links cysteine residues inserted in
the primary sequence. Preliminary SRCD
experiments show folding after photolysis.
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Issues for the development of SRCD
Water absorbance
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Extended wavelength SRCD
Myoglobin in hexafluoro-2-propanol
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Issues for the development of SRCD
Radiation damage
The flux of CD12 is at the limit for CD of
unprotected biological samples. Faster data
collection techniques will be useful.
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Issues for the development of SRCD
Data analysis (1) Conventional CD analysis basis
sets typically contain between 20 and
40 spectra. Minor secondary structure types
such as polyproline II and 310 helix are grossly
under-represented in these basis sets. Analysis
of CD spectra of all proteins using these sets
always yields a small percentage of these
structure types, but is this really
representative of the proteins true secondary
structure content? Basis sets must be larger and
contain a wider range of secondary structure
types.
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Issues for the development of SRCD
Data analysis (2) Accurate knowledge of protein
concentration is critical for accurate
determination of secondary structure content
using standard basis sets. Colorimetric /
absorbance methods are not good enough. Only
quantitative amino acid analysis gives
sufficiently accurate concentrations. But a new
method has recently been described - g-factor
analysis. G-factor spectrum - CD spectrum
divided by absorbance spectrum. Eliminates
protein concentration and cell pathlength
effects. Baker BR,
Garrell RL. Faraday Discuss. 2004126209-22
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Future developments
Fold recognition/proteomics Large numbers of
protein structures are becoming available. This
has the potential for production of much more
extensive basis sets. Classification using
criteria other than simple secondary structure
content may become feasible (e.g. fold
class). The combination of new analysis methods,
automated sample handling, and array detection
may give SRCD an important role in proteomics
programmes.
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Future developments
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Future developments
Energy-dispersive SRCD - Si diode array detector
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Future developments
Ultra-fast CD
Zhang et al., J. Phys. Chem. 97, 5499 (1993)
Requires Highly collimated, high flux light
source in the UV/VUV (best so far Ti-Sapphire
200 nm) Disadvantage Sensitive to artefacts
such as linear dichroism and linear and circular
birefringence
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Future developments
CD microscopy
NIJI-II Polarizing Undulator Switching frequency
2 Hz Beam size at sample 0.7 mm Degree of
circular polarization gt0.95
Yamada et al., Jpn. J. Appl. Phys. 39, 310 (2000)
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Future developments
New sources - ERLs and FELs
120 nm
1.0E22
Cavity FEL
1.0E21
1.0E20
1.0E19
1.0E18
Ti -Sapphire
1.0E17
Average flux (photons/s/0.1)
1.0E16
1.0E15
1.0E14
1.0E13
1
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5
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Photon Energy (eV)