Infrared Spectroscopy Circular Dichroism - EMBL Hamburg · Electromagnetic spectrum: Infrared...
Transcript of Infrared Spectroscopy Circular Dichroism - EMBL Hamburg · Electromagnetic spectrum: Infrared...
Infrared Spectroscopy&
Circular Dichroism
Haydyn Mertens PhD
Infrared Spectroscopy
Electromagnetic spectrum:
Infrared Spectroscopy
Functional groups absorb IR radiationInduced 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
Stretching of bonds (eg. water)
Vibrational modes
Symmetric Asymmetric Bending
3 fundamental vibration modes
Harmonic oscillator simple exampleeg. 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)
Wavenumbernumber of wavelengths (l) per distance
Units for IR spectroscopy
v = 1/l (cm-1)
proportional to frequencyproportional to photon energy
Infrared (IR) Absorption: Proteins
Barth & Zscherp, Quart. Rev. Biophys. 2002, 35(4), 369-430.
Traditional IR spectroscopy "dispersive"Monochromatic beamMeasure absorbanceScan across different wavelengths
FTIRBroadband usedExcite multiple states/modesAdjust broadband and repeat Deconvolute spectrum
Infrared (IR) spectroscopy
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
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
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
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
Limited information available.Lack of spatial information.Spectral congestion
1D FTIR: Proteins
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
fs pulsed spectroscopyFrequency domain (pump-probe)Time domain (echo)
2D-FTIR
fs pulsed spectroscopyCoupled systemsSee coherences
2D-FTIR
Small molecule example: acetyl-acenato-rhodium dicarbonyl (RDC)
2D-FTIR
2D-FTIR: Proteins
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
Characteristic "shapes"Spectral patterns for secondary structure
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
Characteristic "shapes"
Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441
Z-shape Figure-8 Diagonally elongated
Access to fast time-scale:
10-13 s 104 s10-11 s 10-6 s 10-3 s10-9 s
Short-range fluctuations(side-chains, torsion-angles, hydrogen bonds)
Secondary structure formation
Domain folding(tertiary contacts)
Folding/Binding(aggregation)
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
Amide I labeling 13C-16O/18O
Specific labelingShifts absorption band (red-shift)Reduces problem of spectral crowding
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
Measure vibrational modesIdentify secondary structureMonitor protein unfoldingInvestigate conformational change
Summary FTIR
Circular Dichroism
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
Differential Absorption
eL - eR
∆e
+
-
e
Adapted from: Johnson, Ann. Rev. Biophys. Chem. 1988. 17: 145-66.
Protein backbone amidesElectronic absorption (UV)Sensitive to orientation of transition dipoles
amide n ---> pi* (210 nm)pi1 ---> pi* (190 nm)
Sensitive to backbone dihedral anglesthus secondary structure
Amide Chromophores
General scheme of electronic transitions
Amide Chromophores
n
n2pz
Amide ChromophoresTransition dipoles
n
Absorption is modulated by Interactions between transitions:
pi1 ---> pi* coupling between peptide groupsMixing n ---> pi* and pi1 ---> pi* within a peptide groupMixing n ---> pi* and pi1 ---> pi* between peptide groups
Influenced by geometry of peptide backbones --> Secondary structure!!!
Secondary Structure
helix, sheet and "other"Characteristic CD Spectra
MyoglobinConcanavalin Abeta-lactoglobulinType VI collagen
∆�
+
-
The alpha-helix
n ---> pi*
pi1 ---> pi*
pi1 ---> pi*∆�
+
-
The beta-sheet
n ---> pi*
pi1 ---> pi*
pi1 ---> pi*∆�
+
-
"Other" (Random coil)
pi1 ---> pi*
pi1 ---> pi*∆�
+
-
Differential ABS left and right polarised light
Circular Dichroism
www.jascoinc.com
Using database of known structuresCalculate amount of helix/sheet/other
Secondary structure contentFold recognition
More data (ie. VUV region) increases the information content
Information content
Secondary structure content Voltage-gated sodium channelMinimal 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
Contin-LLSelcon 3CDSSTRVARSLCK2dDichroweb server
http://dichroweb.cryst.bbk.ac.uk
Programs