From Gerwert

Mid-Infrared (MIR) (Vibrational) Spectroscopy

Includes infrared absorption spectroscopy (classic IR and FTIR, transmission and reflection) and infrared scattering spectroscopy (Raman)

Absolute

Difference

Relative Complexity of the IR Spectra

From Ernst & Bartl

Infrared (Vibrational) Spectroscopy: Drawbacks and Advantages

• Drawbacks:1. Very complex spectra; IR bands often have contributions

from the vibrations of several groups

2. Hard to assign bands to the individual groups (e.g., a protein may have 10 Asp with similar bands)

3. High absorbance of water is a big problem

4. Expensive and labour-consuming measurements

• Advantages:1. Information is collected on a molecular level

2. Almost any chemical group has IR bands

3. Very environment-sensitive (H-bonding!!)

4. Possible to learn orientation of individual amino acids and waters, not only chromophores

Water Absorbance

Strongly Masks IR

Protein Bands

From Haris et al

Extracting the Information from

IR Spectra: Deconvolution and Derivative

From Arrondo et alSarcoplasmicreticulum

Basics of Classic IR• IR-transparent windows are necessary (CaF2 and BaF2

are the most common ones)• To be active in the IR spectrum, the vibration of the

molecule should change its dipole moment• Major modes of vibration

1. Symmetric stretch - ν1

2. Bending mode - ν2 (scissors, rocks, wags)3. Asymmetric stretch - ν3

• Complications: Combination (e.g., ν1+ν3) and difference (e.g., ν1-ν2) bands, overtones (2ν1), Fermi resonance, intermolecular coupling

• Bond strength – frequency shift relation• Group vibrations and coupling – how to assign?

Assignment of IR Bands

• Normal mode calculations (only for simple molecules)

• Isotope exchange (H/D)

• Isotope labeling (including SDIL)

• Model compounds

• Mutations

• Chemical modifications

Adsorption of Infrared Radiation

Adsorption of Infrared Radiation

Carbon Dioxide Molecule

Carbon Dioxide Molecule

Protein Structure From Infrared Studies

• Bonds demonstrate characteristic oscillation frequency => normal modes of oscillation

• For proteins, normal vibrational modes present within individual residues have been modelled by studying N-methylacetamide (NMA) (see Arrondo et al. reference)– 9 useful bands => Amide A, B, I-VII bands

Schematic diagram of N-methylacetamide

Protein Normal Modes

(a) Amide A and B (NH stretching, arises as a doublet)

(b) Amide I (80% CO stretching, 20% other)

(c) Amide II (60% NH bending, 40% CN stretching)

(d) Amide III

(e) Amide IV

(f) Amide V

(g) Amide VI

(h) Amide VII

(i) 1070 cm-1

(j) 908 cm-1

(k) 498 cm-1

(l) 274 cm-1

Proteins: Secondary Structure

• Theoretically, Amide-A, -I, -II and -III bands are most useful for determining protein structure

• Due to experimental limitations, only Amide-I and -II bands are typically used– Amide-I is most commonly used for determining protein structure

• “Standard” samples are studied to determine characteristic features of spectra produced by secondary structures (e.g. α-helices, β-sheets)– polylysine is often used, as it will adopt random coil, α-helical or β-sheet

conformations depending on physiological conditions

• Secondary structure determined by observing shifts in Amide-Ibands– considering bonds as 2 point masses connected by a spring, hydrogen

bonding will lower the “spring constant” of the bond, resulting in lower vibrational frequency

Proteins: Secondary Structure

STRUCTURE AMIDE-I ν

(cm-1)___

antiparallel β-sheet 1675 - 1695

310-helix 1660 - 1670

α-helix 1648 - 1660

random coil 1640 - 1648

β-sheet 1625 - 1640

aggregated strands 1610 - 1628

Jackson & Mantsch, Crit. Rev. Biochem. Mol. Biol. 30, pp.95-120. (1995)

Arrondo et al., Prog. Biophys. Mol. Biol. 59, pp.23-56. (1992)

IR Vibrational Bands of Water

From Barrow

Major Group Vibrations: Strongly Bound Water

From Kandori

OH stretch – 3200-3500 cm-1; OD stretch – 2400-2600 cm-1; bending ~ 1650 cm-1

Major Group Vibrations: Protein Backbone

• Amide bands reflect global conformational changes• Amides A and B (N-H stretch), 3100 and 3300 cm-1

• Amide I (mostly C=O stretch), 1600-1700 cm-1

Often used to determine protein secondary structure, even though the correlation is not absolute: α-helix is at 1648-1660 cm-1, β-sheet is at 1625-1640 cm-1, turns are at 1660-1685 cm-1, and unordered peptides are at 1652-1660 cm-1

• Amide II (mostly N-H bend and C-N stretch), 1510-1580 cm-1; very sensitive to H/D exchange, so it is often used (along with Amide I) to check solvent accessibility of the protein core and to distinguish between unordered and helical conformations

H/D Exchange of Amide ProtonsFrom Haris et al

chymotrypsin

Amide I – β-sheetAmide II

NH ND

Following Protein Unfolding In the Amide Region

From Haris et al

Citrate synthase in D2O

Amide I –aggregated β-sheet

Amide I –α-helix

Amide I –α-helix

Amide I –aggregated β-sheet

Major Group Vibrations: Protein Side-Chains• Protonation/deprotonation, pKa and environmental changes

(H-bonding) of Asp/Glu (1700-1770 cm-1 for protonated C=O stretch)

• Environmental changes of Asn/Gln (1670-1700 cm-1 for C=O stretch)

• Environmental changes of Tyr (C-O- stretch) at ~ 1500 cm-1

• Environmental changes of Thr (O-H stretch) at 3400-3500 cm-1

• Environmental changes of Trp (N-H stretch) at ~ 3500 cm-1

• Environmental changes of Cys (S-H stretch) at 2400-2600 cm-1

Major Group Vibrations: Lipids

• PO2- stretches (1085 and 1228 cm-1) -

phospholipids

• COO- stretches (1623 cm-1)

• various C-H and CH3 stretches and bends

• N+(CH3)3 bends and stretches – choline-based lipids

Difference spectrum of the N intermediate of the photocycle

N

Data: A. Dioumaev

Typical Characterization of Bacteriorhodopsin by Infrared Spectroscopy

FTIR versus Classic (Dispersive) IR

From Gerwert

Dispersive IR FTIR

FTIR versus Classic (Grating) IR

• Interferometer-based vs. monochromator- based

• Interferogram contains information about all spectral elements (multiplex or Felgett advantage)

• Interferometers have much higher light throughput (Jacquinot advantage)

From Siebert

∫+∞

∞−

= dxxxsS ]2cos[)()( πνν

Fourier Transform Infrared (FTIR) Spectrometry

• Michelson interferometer is the heart of most commercially available FTIR spectrometers– as movable mirror is swept from left to right, optical path difference

between light in the two arms of the interferometer increases– for a single wavelength IR source, constructive and destructive

interference conditions are periodically observed

Fourier Transform IR spectroscopy allows for multiple IR wavelengths to be measured simultaneously!

FTIR Spectra

• Tens to hundreds of interferograms are collected and averaged

• Inverse Fast Fourier Transform (FFT) of interferogram produces transmission spectrum

• Spectra measured with and without sample in beamline are compared– transmittance τ = I/Io

– transmission T = -log10τ

FTIR: Benefits & Pitfalls

Water absorbs strongly in the IR region of the spectrum Absorption bands often overlap, necessitating complicated

deconvolution routines Fourier self deconvolution, derivative techniques, least squares fitting

No information regarding positional structure! for known structures, can use FTIR to monitor changes in

conformation

Non-destructive technique

Can be performed on samples with any morphology (crystals, membrane-bound, gels, etc.)

Small sample volumes (10 mL of protein solution, even less for some variations of FTIR)

Extremely good time resolution (as low as 30 ns)

Simple Scheme of FTIR Spectrometer

From Siebert

Simple Scheme of FTIR Spectrometer

From Kotting et al

Bruker

Simple Scheme of FTIR Spectrometer

Bruker FTIR spectrometer layout

IR Sample Holders

From Kotting et al

Transmission

Reflection

Flow

Difference FTIR Spectroscopy

Can be either static (photostationary or low-temperature) or time-resolved

From Siebert

Absolute vs. Difference FTIR of Ras

Protein

From Kotting et al

Difference Spectra of Carboxylic Acids

From Nyquist et al

Time-Resolved (Reaction-Induced) Difference FTIR

Methods of Triggering:1. Light

2. Caged compounds

3. Mixing

From Gerwert

Caged Phosphate

Time-Resolved (Reaction-Induced) FTIR

Artwork: Lichi Shi

Time-Resolved Difference FTIR

Artwork: Lichi Shi

Time-Resolved Difference FTIR

From Siebert

Two Major Kinds of Time-Resolved FTIR

• Rapid-scan1. One set of interferograms is scanned per each flash2. Can not go faster than 10 ms per spectrum3. Relatively low noise, fast results

• Step-scan1. One time-slice is measured for each position of the

moving mirror, then the spectra are reconstructed2. High time-resolution (up to ns)3. Relatively high-noise, very time-consuming, requires

fast detectors and A/D converters

Time-Resolved FTIR of Noncyclic Reactions

From Gerwert

Some Important Transition Dipole Moments of Lipids and Membrane Proteins

From Tamm et al

Attenuated Total Reflection (ATR) FTIR1. Overcomes the

problem of water absorbance

2. Allows titration of the sample without disturbing it

3. Convenient for measurements of molecular orientations

4. Diamond ATR can be used to measure just a few microliters of the sample

Crystal is usually KRS, ZnSe, Ge or diamond

From Siebert

Micro-ATR (diamond)

From Siebert

Rugged, small, optically superior

ATR-IR Titration of Carboxylic

Acids(Asp, Glu)

Citrate

COOH

COO-

From Goormaghtigh et al

Molecular Orientations:

Transmission vs. ATRFTIR

From Tamm et al

Polarized ATR-FTIR To Determine

Lipid Orientation and Order

From Tamm et al

DMPC C-H

NIR Spectroscopy as a Promising Biomonitoring Tool

• NIR region (800-2500 nm) contains only weak bands due to overtones and combinations of fundamental vibrations

• Can be used for in situ monitoring of media, fibers, etc. (weak signals allow measurements of high concentrations and long pathlength)

• Also much cheaper than MIR (glass optics, conventional light sources and detectors)

Raman Spectroscopy: Basics• Inelastically scattered radiation collected at 90° (or 180°) to the

monochromatic light source (UV, visible, or near IR)

From Browne et al

Raman Spectroscopy: Basics

• Rayleigh (elastic) scattering ν0, Stokes lines ν0-νv, anti-Stokes lines ν0+νv

From Browne et al

Raman Spectroscopy: Basics• The scattering is very inefficient, so powerful lasers are needed.

This sometimes creates a problem of the photostationary mixtures and sample damage. Does not have a problem with water absorbance

From Browne et al

Raman Spectroscopy: Basics• Different from IR selection rules – there must be a polarizability

change during the vibration. Thus, Raman can give additional information through the lines which are not active in classic IR (D=αE)

From Miura et al

Example of Complementarity of IR and Raman

Resonance Raman

From Browne et al

From Koechendorfer

Raman Bands of the Chromophore of Rhodopsins

11-cis-retinal

Raman Titration of SH groups (Cys)

From Miura et al

thioredoxin

Specialized Raman Techniques in Biophysics

• FT-Raman (no problem of electronic excitation, photobleaching, and fluorescence)

• UV-Raman• Time-resolved (kinetic) Raman1. Pump+Probe2. Spinning Cells3. Flow cells• Surface-enhanced Raman (silver electrodes and

hydrosols) - SERS• Raman Microscopes (Spectra+Imaging)• CARS – coherent anti-Stokes Raman Spectroscopy

FT-Raman Spectra of Microbial Rhodopsins

UV-Raman –Following

Aromatic Amino Acids and

Nucleotides

From Thomas

Pump+Probe Resonance

Raman

From Eisfeld et al

A New Word in Raman – Stimulated Raman

From Kukura et al

Femtosecond Stimulated Raman –

Probing Fast Photobiological

Reactions

From Kukura et al

Application of Femtosecond

Stimulated Raman to Vision

From Kukura et al

Example of Carboxylic Acid

Bands Assignment Using Mutagenesis and

H/D Exchange

Proteorhodopsin

H

D

Fourier Transform infrared spectroscopy

0 1000 2000 3000 4000

0

1

2

50 0 -50-0.5

0.0

0.5

2000 1500 1000 500 0-0.5

0.0

0.5

Infra

red

light

Inte

nsity

(A.U

.)

Inte

rfero

gram

Inte

nsity

(A.U

.)

δ(4xλHe-Ne/2)ν(cm-1)

A

B

Bruker IFS66v/S manual

Transmission Polarized FTIR to Determine Orientation of Various Molecular Groups

Inside the Protein

From Hatanaka et al

C=O Asp85

C=O Asp85