Absolute
Difference
From Gerwert
Mid-Infrared (MIR) (Vibrational) Spectroscopy Includes infrared absorption spectroscopy (classic IR and FTIR, transmission and reflection) and infrared scattering spectroscopy (Raman)
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. 2. 3. 4.
Information is collected on a molecular level Almost any chemical group has IR bands Very environment-sensitive (H-bonding!!) Possible to learn orientation of individual amino acids and waters, not only chromophores
From Haris et al
Water Absorbance Strongly Masks IR Protein Bands
From Arrondo et al Sarcoplasmic reticulum
Extracting the Information from IR Spectra: Deconvolution and Derivative
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 Nmethylacetamide (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. αhelices, 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-I bands – 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 Protons From Haris et al chymotrypsin
Amide I – β-sheet
Amide II NH
ND
Following Protein Unfolding In the Amide Region Amide I – α-helix
Citrate synthase in D2O Amide I – aggregated β-sheet
From Haris et al
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
Typical Characterization of Bacteriorhodopsin by Infrared Spectroscopy
N
Data: A. Dioumaev
Difference spectrum of the N intermediate of the photocycle
FTIR versus Classic (Dispersive) IR FTIR
Dispersive IR
From Gerwert
FTIR versus Classic (Grating) IR
From Siebert
S (ν ) =
+∞
∫ s( x ) cos[2πνx ]dx
−∞
• Interferometer-based vs. monochromator- based • Interferogram contains information about all spectral elements (multiplex or Felgett advantage) • Interferometers have much higher light throughput (Jacquinot advantage)
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 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) 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
Simple Scheme of FTIR Spectrometer
From Siebert
Simple Scheme of FTIR Spectrometer
Bruker
From Kotting et al
Simple Scheme of FTIR Spectrometer
Bruker FTIR spectrometer layout
IR Sample Holders Transmission
Flow
Reflection
From Kotting et al
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-scan
1. One set of interferograms is scanned per each flash 2. Can not go faster than 10 ms per spectrum 3. Relatively low noise, fast results
•
Step-scan
1. One time-slice is measured for each position of the moving mirror, then the spectra are reconstructed 2. 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) FTIR 1. Crystal is usually KRS, ZnSe, Ge or diamond
2.
3.
4. From Siebert
Overcomes the problem of water absorbance Allows titration of the sample without disturbing it Convenient for measurements of molecular orientations Diamond ATR can be used to measure just a few microliters of the sample
Micro-ATR (diamond) Rugged, small, optically superior
From Siebert
COO-
COOH
ATR-IR Titration of Carboxylic Acids (Asp, Glu)
Citrate
From Goormaghtigh et al
Molecular Orientations: Transmission vs. ATR FTIR From Tamm et al
DMPC C-H
From Tamm et al
Polarized ATR-FTIR To Determine Lipid Orientation and Order
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) Example of Complementarity of IR and Raman
From Miura et al
Resonance Raman
From Browne et al
Raman Bands of the Chromophore of Rhodopsins 11-cis-retinal
From Koechendorfer
Raman Titration of SH groups (Cys)
thioredoxin
From Miura et al
Specialized Raman Techniques in Biophysics FT-Raman (no problem of electronic excitation, photobleaching, and fluorescence) • UV-Raman • Time-resolved (kinetic) Raman 1. Pump+Probe 2. Spinning Cells 3. 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
Proteorhodopsin
H D
Example of Carboxylic Acid Bands Assignment Using Mutagenesis and H/D Exchange
Fourier Transform infrared spectroscopy 0.5
1
B
Interferogram Intensity (A.U.)
Infrared light Intensity (A.U.)
2
0.5
0.0
0.0
-0.5 50
0
-50
A
0 0
1000
2000
ν(cm-1)
3000
4000
-0.5 2000 1500 1000
500
0
δ(4xλHe-Ne/2)
Bruker IFS66v/S manual
Transmission Polarized FTIR to Determine Orientation of Various Molecular Groups Inside the Protein
C=O Asp85
From Hatanaka et al
C=O Asp85
