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