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
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
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
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)
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
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 (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)
Difference FTIR Spectroscopy
Can be either static (photostationary or low-temperature) or time-resolved
From Siebert
Time-Resolved (Reaction-Induced) Difference FTIR
Methods of Triggering:1. Light
2. Caged compounds
3. Mixing
From Gerwert
Caged Phosphate
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
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
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
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
Fourier Transform infrared spectroscopy
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δ(4xλHe-Ne/2)ν(cm-1)
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Bruker IFS66v/S manual