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Infrared Spectroscopy & Circular Dichroism Haydyn Mertens PhD - - PowerPoint PPT Presentation
Infrared Spectroscopy & Circular Dichroism Haydyn Mertens PhD - - PowerPoint PPT Presentation
Infrared Spectroscopy & Circular Dichroism Haydyn Mertens PhD Infrared Spectroscopy Infrared Spectroscopy Electromagnetic spectrum: VIS med long-wave IR Short Gamma X-Ray UV IR microwaves radiowaves nm um mm m km Wavelength
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Electromagnetic spectrum:
Infrared Spectroscopy
Functional groups absorb IR radiation Induced vibrational excitation
Wavelength 10-9 10-6 10-3 1 10 102 m nm um mm m km Gamma X-Ray UV IR microwaves
Short
radiowaves VIS med long-wave IR
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Stretching of bonds (eg. water)
Vibrational modes
Symmetric Asymmetric Bending
3 fundamental vibration modes
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Harmonic oscillator simple example
- eg. diatomic molecule
Vibrational modes
Vibrational frequency: v ∝ k*mr
eg. C=O, C=N (1500 - 1900 cm-1) C-H, N-H, O-H (2700 - 3800 cm-1)
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Wavenumber number of wavelengths (l) per distance
Units for IR spectroscopy
v = 1/l (cm-1)
proportional to frequency proportional to photon energy
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Infrared (IR) Absorption: Proteins
Barth & Zscherp, Quart. Rev. Biophys. 2002, 35(4), 369-430.
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Traditional IR spectroscopy "dispersive" Monochromatic beam Measure absorbance Scan across different wavelengths FTIR Broadband used Excite multiple states/modes Adjust broadband and repeat Deconvolute spectrum
Infrared (IR) spectroscopy
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Key component is the interferometer
FTIR spectroscopy
Detected signal: Intensity as function of mirror position (cm) FT to obtain IR spectrum (cm-1)
http://www.chem.agilent.com
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Sensitive to secondary structure:
1D FTIR: Proteins
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
Amide I and Amide II modes
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Sensitive to secondary structure:
1D FTIR: Proteins
Adapted from: Barth & Zscherp, Quart. Rev. Biophys., 2002, 35, pp 369-430
Amide I and Amide II modes
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Sensitive to secondary structure:
1D FTIR: Proteins
Adapted from: Barth & Zscherp, Quart. Rev. Biophys., 2002, 35, pp 369-430
Amide I and Amide II modes
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Limited information available. Lack of spatial information. Spectral congestion
1D FTIR: Proteins
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
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fs pulsed spectroscopy Frequency domain (pump-probe) Time domain (echo)
2D-FTIR
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fs pulsed spectroscopy Coupled systems See coherences
2D-FTIR
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Small molecule example: acetyl-acenato-rhodium dicarbonyl (RDC)
2D-FTIR
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2D-FTIR: Proteins
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
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Characteristic "shapes"
Spectral patterns for secondary structure
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
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Characteristic "shapes"
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
Z-shape Figure-8
Diagonally elongated
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Access to fast time-scale:
10-13 s 104 s 10-11 s 10-6 s 10-3 s 10-9 s
Short-range fluctuations
(side-chains, torsion-angles, hydrogen bonds)
Secondary structure formation Domain folding (tertiary contacts) Folding/Binding (aggregation)
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Thermal denaturation of Ubiquitin
(Chung et al., PNAS. 2007, 104, 14237-14242.)
Example: Protein Unfolding
2D FTIR from MD simulation Experiment
Folded Unfolded Difference
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
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Amide I labeling 13C-16O/18O
Specific labeling Shifts absorption band (red-shift) Reduces problem of spectral crowding
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M2 (influenza A), H+ gated ion channel Transmembrane helix conformation
13C=18O labeled residues as probes
Example: Membrane Protein
Manor et al., Structure. 2009, 17, pp 247-254.
Linewidth 13C=18O increases with solvent contact
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Measure vibrational modes Identify secondary structure Monitor protein unfolding Investigate conformational change
Summary FTIR
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Circular Dichroism
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Absorbance spectroscopy of electronic transitions: A = e*c*l
e = extinction coefficient (depends on wavelength, l) c = concentration l = path length
CD is difference between e for left and right circularly polarized light
AL(l) - AR(l) = ∆A(l) = [eL(l) - eR(l)]*l*c
Circular Dichroism
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Differential Absorption
eL - eR ∆e +
- e
Adapted from: Johnson, Ann. Rev. Biophys. Chem. 1988. 17: 145-66.
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Protein backbone amides Electronic absorption (UV) Sensitive to orientation of transition dipoles amide n ---> pi* (210 nm) pi1 ---> pi* (190 nm) Sensitive to backbone dihedral angles thus secondary structure
Amide Chromophores
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General scheme of electronic transitions
Amide Chromophores
n n 2pz
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Amide Chromophores
Transition dipoles n
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Absorption is modulated by Interactions between transitions:
pi1 ---> pi* coupling between peptide groups Mixing n ---> pi* and pi1 ---> pi* within a peptide group Mixing n ---> pi* and pi1 ---> pi* between peptide groups
Influenced by geometry of peptide backbones --> Secondary structure!!!
Secondary Structure
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helix, sheet and "other"
Characteristic CD Spectra
Myoglobin Concanavalin A beta-lactoglobulin Type VI collagen
∆ +
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The alpha-helix
n ---> pi* pi1 ---> pi* pi1 ---> pi*
∆ +
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The beta-sheet
n ---> pi* pi1 ---> pi* pi1 ---> pi*
∆ +
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"Other" (Random coil)
pi1 ---> pi*
pi1 ---> pi*
∆ +
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Differential ABS left and right polarised light
Circular Dichroism
www.jascoinc.com
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Using database of known structures Calculate amount of helix/sheet/other Secondary structure content Fold recognition More data (ie. VUV region) increases the information content
Information content
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Secondary structure content Voltage-gated sodium channel Minimal functional tetramer designed
Example:
McCusker et al., J. Biol. Chem. 2011, March 15 (epub)
CD sprectrum (50 % helix) Melting curve (222 nm)
30% helix 19% helix
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