Fourier Transform Infrared (FTIR) Spectroscopy


Fourier transform infrared (FTIR) spectroscopy is an experimental technique used initially for qualitative and quantitative analysis of organic compounds, providing specific information on molecular structure, chemical bonding and molecular environment. For many years, FTIR has been successfully employed for studying a wide variety of proteins, enzymes, nucleic acids, lipids and glycolipids and photobiological systems. Time‐resolved (tr)‐FTIR spectroscopy can monitor reactions of the amino acids, the ligands and specific water molecules in the active centre of a protein in the time range from nanoseconds to seconds, thereby providing a detailed understanding of the molecular reaction mechanism.

Key Concepts:

  • FTIR provides several advantages compared to other spectroscopic techniques.

  • FTIR spectroscopy is widely used for the investigation of secondary protein structure.

  • To obtain information on individual groups, we have to look at difference spectra.

  • In order to derive information on the reaction mechanism, the infrared bands have to be assigned to molecular groups of the protein.

  • The QM/MM (quantum mechanics/molecular mechanics) approach can be used to calculate the infrared spectra of proteins.

  • With the ATR (attenuated total reflection) technique it is possible to immobilise a protein on a surface and study reactions with ligands or other proteins.

  • Biochemical changes of tissues and body fluids can be monitored using FTIR.

  • By means of infrared microscopes spatial resolved information with a resolution of a few micrometer can be obtained.

Keywords: secondary protein structure; lipid characterisation; pKa determination; medicinal biology; proteins; molecular reaction mechanism; time‐resolved spectroscopy

Figure 1.

(a) Typical setup of a Fourier transform infrared (FTIR) spectrometer: the light from an infrared light source is sent through an aperture hole to a mirror (the ‘beamsplitter’) that sends two equivalent beams (one reflected and one transmitted) to a fixed and to a scanning retroreflector mirror, respectively. After acquiring different optical path lengths (in addition to the equal distances of both mirrors from the beamsplitter – the ‘zero path difference’) these two beams recombine on the beamsplitter and are sent to the sample, and after this to a semiconductor detector. The detector signal as function of the optical path difference between the two mirrors is the interferogram (b). After fast Fourier transform (FFT) calculation the infrared spectrum is obtained. Reproduced from Kötting C and Gerwert K (2007) Protein reactions: resolved with tr‐FTIR. Spectroscopy Europe19(3): S19–S23.

Figure 2.

Typical absorbance spectrum of a protein solution (here 10 mM Ras). The main components are indicated by the colours red (CO stretching vibration, amide I), blue (bending vibration, water) and green (combination of NH bending and CN stretching vibration, amide II). The amide I band is decomposed for the secondary structure. In the inset, two absorption spectra of a protein, which deviate only in the protonation of a carboxyl group, are shown schematically. In the lower part, a difference spectrum of these two states is shown schematically. The background absorptions of the unchanged part of the protein are cancelled out; the absorptions of the reacting group are now resolved. Reproduced from Kötting C and Gerwert K (2007) Protein reactions: resolved with tr‐FTIR. Spectroscopy Europe19(3): S19–S23.

Figure 3.

(a) Band assignment by site‐directed mutagenesis. In the difference spectrum N–BR of the wild‐type (WT) (blue), the positive band shows the protonation of Asp85, the negative band shows the deprotonation of Asp96 and the difference band shows the change in the hydrogen bonding of Asp115. The negative band of Asp96 disappears in the Asp96Asn mutant (red). The difference band of Asp115 disappears in the Asp115Asn mutant (green). (b) Band assignment by isotopic labelling: In the hydrolysis spectrum of Ras, the bands facing downwards are from the Ras·GTP state (see, and; the bands facing upwards are from the Ras·GDP state. Black: unlabelled; green: γ‐18O3‐GTP. Owing to the labelling, the absorption of the νas(γ‐PO3)‐vibration has shifted, whereas the other bands, for example the absorption of the νas(α‐PO2)‐vibration are unchanged. The bands of the released Pi facing upwards at 1078 and 990 cm−1 are also shifted by the label. Below, the calculated normal modes of the isolated νas(γ‐PO3)‐vibration are shown. Reproduced from Kötting C and Gerwert K (2007) Protein reactions: resolved with tr‐FTIR. Spectroscopy Europe19(3): S19–S23.

Figure 4.

(a) Time‐dependent absorbance changes during the bR photocycle from 30 ns to 200 ms at 4 cm−1 resolution. The absorbance of protonated Asp85 (1762 cm−1) and protonated Schiff base (PSB) (1190 cm−1) are indicated. In the L→M reaction (completed after 60 μs), a proton is transferred from the PSB to Asp85, as seen in the decay at 1190 cm−1 and the increase at 1762 cm−1. (b) Owing to light‐induced isomerisation of the chromophor retinal, the strong H‐bond of water 402 is broken and approximately half of the energy is stored in the protein. After isomerisation, the free OH‐group of the dangling water 401 is H‐bonded and can no longer stabilise the charge of Asp85. The proton is transferred from the PSB to Asp85. Owing to the neutralisation of Asp85, a downward movement of Arg82 is induced. This movement of the positive charge destabilises the protonated water complex near the protein surface. The protonated water cluster (blue) stores a proton, probably in an asymmetric Eigen‐complex (H+(H2O)3). This destabilises the second hydration shell. In contrast to the random Grotthuss‐proton transfer in water, in the protein the water complex is deprotonated by a directed movement of Arg82. The proton is stabilised in the second hydration shell by amino acids instead of water molecules. Reproduced from Kötting C and Gerwert K (2007) Protein reactions: resolved with tr‐FTIR. Spectroscopy Europe19(3): S19–S23.



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Further Reading

Barth A and Haris PI (2009) Biological and Biomedical Infrared Spectroscopy, Advances in Biomedical Spectroscopy, vol. 2. Amsterdam, The Netherlands: IOS Press.

Diem M, Griffiths P and Chalmers J (2008) Vibrational Spectroscopy for Medical Diagnosis. New York: Wiley.

Griffiths PR and de Haseth JA (2007) Fourier Transform Infrared Spectrometry, 2nd edn. Hoboken, NJ: Wiley.

Kötting C and Gerwert K (2005) Monitoring protein‐protein interactions by time‐resolved FTIR difference spectroscopy. In: Golemis EA and Adams PD (eds) Protein–Protein Interactions, 2nd Edition, pp. 279–299. Cold Spring Harbor, NY: CSHL press.

Siebert F and Hildebrandt P (2007) Vibrational Spectroscopy in Life Science. Weinheim, Germany: Wiley‐VCH.

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Gerwert, Klaus, and Kötting, Carsten(Sep 2010) Fourier Transform Infrared (FTIR) Spectroscopy. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0003112.pub2]