Infrared Spectroscopy & Circular Dichroism

Haydyn Mertens PhD Infrared Spectroscopy Infrared Spectroscopy

Electromagnetic spectrum:

VIS Short med long-wave IR

Gamma X-Ray UV IR microwaves radiowaves

nm um mm m km

-9 -6 -3 Wavelength 10 10 10 1 10 102 m Functional groups absorb IR radiation Induced vibrational excitation Vibrational modes

Stretching of bonds (eg. water)

Symmetric Asymmetric Bending

3 fundamental vibration modes Vibrational modes Harmonic oscillator simple example eg. diatomic molecule

Vibrational frequency: ∝ k*m v r

eg. C=O, C=N (1500 - 1900 cm-1) C-H, N-H, O-H (2700 - 3800 cm-1) Units for IR spectroscopy

Wavenumber number of wavelengths (l) per distance

v = 1/l (cm-1)

proportional to frequency proportional to photon energy Infrared (IR) Absorption: Proteins

Barth & Zscherp, Quart. Rev. Biophys. 2002, 35(4), 369-430. Infrared (IR) spectroscopy

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 FTIR spectroscopy Key component is the interferometer

http://www.chem.agilent.com Detected signal: Intensity as function of mirror position (cm) FT to obtain IR spectrum (cm-1) 1D FTIR: Proteins

Sensitive to secondary structure:

Amide I and Amide II modes

Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441 1D FTIR: Proteins

Sensitive to secondary structure:

Amide I and Amide II modes

Adapted from: Barth & Zscherp, Quart. Rev. Biophys., 2002, 35, pp 369-430 1D FTIR: Proteins

Sensitive to secondary structure:

Amide I and Amide II modes

Adapted from: Barth & Zscherp, Quart. Rev. Biophys., 2002, 35, pp 369-430 1D FTIR: Proteins

Limited information available. Lack of spatial information. Spectral congestion

Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441 2D-FTIR fs pulsed spectroscopy Frequency domain (pump-probe) Time domain (echo) 2D-FTIR fs pulsed spectroscopy Coupled systems See coherences 2D-FTIR Small molecule example: acetyl-acenato-rhodium dicarbonyl (RDC) 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"

Diagonally Z-shape Figure-8 elongated

Ganim et al., Acc. Chem. Res., 2008, 41 (3), pp 432–441 Access to fast time-scale:

10-13 s 10-11 s 10-9 s 10-6 s 10-3 s 104 s

Short-range fluctuations Secondary structure Domain folding Folding/Binding (side-chains, torsion-angles, formation (tertiary contacts) (aggregation) hydrogen bonds) Example: Protein Unfolding

Thermal denaturation of Ubiquitin (Chung et al., PNAS. 2007, 104, 14237-14242.)

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 labeling Shifts absorption band (red-shift) Reduces problem of spectral crowding Example: Membrane Protein M2 (influenza A), H+ gated ion channel Transmembrane helix conformation 13C=18O labeled residues as probes

Linewidth 13C=18O increases with solvent contact Manor et al., Structure. 2009, 17, pp 247-254. Summary FTIR Measure vibrational modes Identify secondary structure Monitor protein unfolding Investigate conformational change Circular Dichroism 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 Differential Absorption

+

e ∆e

- eL - eR

Adapted from: Johnson, Ann. Rev. Biophys. Chem. 1988. 17: 145-66. Amide Chromophores 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 General scheme of electronic transitions

2pz n

n Amide Chromophores Transition dipoles

n Secondary Structure 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!!! Characteristic CD Spectra helix, sheet and "other"

Myoglobin Concanavalin A + beta-lactoglobulin Type VI collagen ∆

- The alpha-helix

pi1 ---> pi*

+

∆

pi1 ---> pi* - n ---> pi* The beta-sheet

+ pi1 ---> pi* ∆ n ---> pi* pi1 ---> pi* - "Other" (Random coil)

+ pi ---> pi* ∆ 1

- pi1 ---> pi* Circular Dichroism Differential ABS left and right polarised light

www.jascoinc.com Information content 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 Example: Secondary structure content Voltage-gated sodium channel Minimal functional tetramer designed CD sprectrum (50 % helix) Melting curve (222 nm) 30% helix

19% helix

McCusker et al., J. Biol. Chem. 2011, March 15 (epub) Programs Contin-LL Selcon 3 CDSSTR VARSLC K2d Dichroweb server http://dichroweb.cryst.bbk.ac.uk