Timeresolved circular dichroism: Application to the study of conformational changes in proteins

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Time-resolved circular dichroismApplication to
the study of conformational changes in proteins

• François Hache
• Laboratoire d'Optique et Biosciences
• CNRS/INSERM/Ecole Polytechnique

PhD students Thibault Dartigalongue Claire
Niezborala
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Conformation of biological molecules

• Many biological processes are triggered by a
  conformational change (allosteric effect in
  hemoglobin, membrane receptors, vision, )
• Enzymatic activity depends on the protein
  structure
• folding process ?
• effects of misfolding (amyloid fibrils)

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Study of the conformational dynamics in
biological processes

• Trigger
• Stopped-flow method (ms)
• T-jump (ns)
• Laser photolysis (fs)

• Conformational probes
• Fluorescence (quenching, FRET)
• Raman
• IR absorption (amide bands)
• Circular dichroism

M. Volk, Eur. J. Org. Chem. 2001, 2605
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Outline

• Time-resolved circular dichroism
• Photodissociation of carbonmonoxy-myoglobin
• experiment
• calculation and discussion
• Extension to the far-UV towards the study of
  protein folding

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Outline

• Time-resolved circular dichroism
• Photodissociation of carbonmonoxy-myoglobin
• experiment
• calculation and discussion
• Extension to the far-UV towards the study of
  protein folding

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Circular dichroism
CD 1/1000, 1/10000
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Origin of the optical activity

• asymmetric carbon
• coupling of nonchiral chromophores

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CD ? conformation change

• changes in the distance, angle, coupling
• change in the rotational strength
• change in the CD

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Pump/probe experiment
Principle
- the pump initiates a reaction
PROBE
detector
PUMP
- the probe measures the transmission of the
excited molecules
variable delay
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Time-Resolved Circular Dichroism
the probe measures the sample CD after excitation
Possibility to follow ultrarapid conformational
changes
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Outline

• Time-resolved circular dichroism
• Photodissociation of carbonmonoxy-myoglobin
• experiment
• calculation and discussion
• Extension to the far-UV towards the study of
  protein folding

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Myoglobin (Mb)
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Photodissociation of MbCO
Carboxymyoglobin (MbCO)
Myoglobin (Mb)
Kachalova, Science (1999)
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CD experiment in the Soret band
Soret band p ? p transition in the heme

• in the visible
• isolated
• ultrarapid electronic relaxation

Strong CD structure
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Experimental set-up
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Problem of artifact
Simon et al., J. Phys. Chem. (1992)
Left- handed probe
Right- handed probe
Pump


pump-induced birefringence

• very stringent alignment procedure for the
  Pockels cell
• T. Dartigalongue and F. Hache, J. Opt. Soc. Am.
  B 20, 1780 (2003)

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Nanosecond timescale
MbCO
CD
Mb
Wavelength (nm)
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Picosecond timescale
Biphasic dynamics 7 ps, 50 ps
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Outline

• Time-resolved circular dichroism
• Photodissociation of carbonmonoxy-myoglobin
• experiment
• calculation and discussion
• Extension to the far-UV towards the study of
  protein folding

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Origin of the CD in myoglobin
M. C. Hsu and R. W. Woody, J. Am. Chem. Soc. 93,
3515-3525 (1971).
Coupled oscillators
Heme absorption in the Soret band (420 nm)
Transition in the near Ultra Violet
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Applequist's polarizability theory
Several chromophores, subscript i
In each chromophore, several optical transitions,
subscript s
N normal modes
N dipoles i.s
J. Applequist, J. Chem. Phys. 58, 4251-4259
(1973). F. Hache et T. Dartigalongue J. Chem.
Phys. 123, 184901 (2005)
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Myoglobin parameters

• Protein structure PDB
• MbCO 1A6G
• Mb 1A6N

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The important aromatic residues
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Rotation of the proximal Histidine

80
70
60
MbCO
50
40
CD
Mb
30

20
10
0


-10

400
450
500
Wavelength (nm)
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Perutz' scenario
1/ photodissociation of MbCO, heme doming 2/
constraint on the proximal Histidine 3/relaxation
towards the deoxy-form
Perutz et al. Acc. Chem. Res. 20, 309 (1987)
Max Perutz Nobel price in chemistry in 1962 for
the hemoglobin structure
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Outline

• Time-resolved circular dichroism
• Photodissociation of carbonmonoxy-myoglobin
• experiment
• calculation and discussion
• Extension to the far-UV towards the study of
  protein folding

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Far-UV CD secondary structure
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UV SOURCE (Thibault Dartigalongue)
490 nm 700 nm
220 nm 350 nm
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Measurement of the CD with a Babinet Soleil
compensator

C
left
Circular dichroism ?0 (aleft aright)L
Optical rotation d0 (2p/?)(nleft nright)L
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Static CD measurement

• IAf(d,?,f)
• without sample d?0
• IA4sin2f f2
• with a chiral sample
• IA (f?/4)2

f retardation
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Measurement of dCD
with the pump ??0d? et aa0da
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Advantages

• Shift of the LI and PM parabolas
• d?/4daL.
• Increased sensitivity thanks to the
• modulation of the pump intensity
• Linearly-polarized probe
• no more artifacts

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Experimental demo RuTP

• Ruthenium-tris(phenanthroline)
• Probe 265nm
• ?02.40.1x10-2
• soit ?e 700M-1cm-1
• daL3.3x10-2
• d?? (2.8 0.7 )x10-3
• d?? (3.8 0.7 )x10-3

C. Niezborala and F. Hache, J. Opt. Soc. Am. B
23, 2418 (2006).
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Photodissociation of MbCO in the UV
CD
C. Niezborala, T. Dartigalongue and F. Hache,
Phys. Chem. Chem. Phys. (2007)
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Folding of a-helices
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Photoinduction of folding ?
? cis-trans isomerization in azobenzene
? disulfide bridge
J. Wachtveitl, W. Zinth. Bioph. J. 86, 2350
(2004)
M. Volk, W.F. DeGrado, R.M. Hochstrasser, J.
Phys. Chem. B 101, 8607 (1997)
? pH-jump
T. Causgrove, R. B. Dyer, Chem. Phys. 323, 2
(2006)
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• Time-resolved Circular Dichroism
• ? ultrafast conformational changes
• Complementary technique to ultrafast Raman or IR
  spectroscopy
• Applicable to 4GLS (especially in the far-UV
  160-240 nm)