Using FTIR Spectroscopy to Determine Protein Secondary Structure


Proteins are the basic building blocks of life. They play a critical role in cell function and structure and are involved in transport, communication  and metabolism in the body. While just 20 amino acids are used to form proteins, their potential structures, sequences, and combinations are almost infinite

Each and every protein fulfills a certain purpose, which often depends on its 3D structure. Defining the 3D structure of proteins is therefore important for gaining insights into how they work, and can provide information on how proteins interact with other molecules, how they function as enzymes and how they undergo conformational changes. This data can be extremely useful in drug design and in the manufacture of industrial enzymes.

High-Resolution Approaches

The method that provides the most comprehensive information on protein structure is X-ray crystallography. However, its application can be limited by the fact that proteins do not always form crystals or that they may form crystals that do not resemble the natural or native state of the protein.

Another high-resolution method is nuclear magnetic resonance (NMR) spectroscopy, which can be used to characterize protein structure in liquids. However, this method is only practical when studying small proteins, measuring 15 to 25kDa. Furthermore, both of these techniques require specific sample preparation steps and specialized skills and can, therefore, take up considerable amounts of time.

A useful alternative approach that can be used to determine secondary protein structure is Fourier Transform Infrared (FTIR) spectroscopy. This technique enables the acquisition of spectra in a vast range of environments and across various different protein sizes. FTIR spectroscopy is also less time consuming, requiring less sample preparation than other techniques.

Insights into Structure

In typical FTIR spectroscopy, infrared light is shone onto a sample and measurements are made of how specific wavelengths are reflected or absorbed. Characteristic absorption bands arise from different structural regions in the protein and this information can be interpreted to determine the secondary structure. This secondary structure, of which the beta-sheet and alpha-helix form the main components, is the most vital aspect of a protein's structure.

The amide I bands derived using FTIR spectroscopy provide information on C=O bonding, while the amide II bands provide information on N-H bonding. This enables important insights about the secondary structure to be gained, since both of these forms of bonding are influenced by the protein’s secondary structural content. Amide I bands, in particular, are very sensitive to this secondary structure.

In addition, FTIR spectroscopy can enable information on proteins’ structural dynamics to be obtained. Protein activity can be affected by the structural dynamics of a specific conformation and these dynamics are essential to the protein performing its function.

Information on structural dynamics is obtained by assessing the rate of exchange of hydrogen isotopes (e.g.deuterium) in water with hydrogen atoms in the protein. This shows the accessibility of hydrogen within N-H bonds in the protein’s 3D structure; a faster rate indicates more motion and flexibility in the protein region undergoing exchange.

FTIR spectroscopy can also be used to study the stability of proteins, including the folding and unfolding processes under various thermal and chemical conditions and also the protein aggregation process, which has been widely studied in Huntington's, Parkinson's, Alzheimer's, and other amyloid diseases. Furthermore, FTIR spectroscopy provides information on protein side–chains - the sequence of R groups that belong to and distinguish the amino acids present in a polypeptide chain.

Although FTIR spectroscopy cannot offer the high-resolution data provided by other methods such as NMR and crystallography, it can quickly and efficiently screen proteins in their native state for a fraction of the cost of these other methods.

A Vital Accessory

The main benefit of FTIR over other methods is convenience. This convenience factor is taken to the next level by Specac's PearlTM accessory, which can be used with any spectrometer available on the commercial market. The special Oyster Cell design enables easy addition of liquid samples to the machine, without any preparation by injection or spotting being needed. The unique design also means cleaning is equally as easy.

Specac's PearlTM is also faster and more reliable than traditional liquid cells, saving both money and time. It can also be used to study highly viscous liquids that cannot be handled by traditional analyzers. Finally, the Specac’s PearlTM provides highly precise and repeatable pathlengths, including the wedging option to prevent fringing, along with interchangeable windows to accommodate various different pathlengths.

References and Further Reading

  • Gallagher W. FTIR Analysis of Protein Structure. Available at: Accessed: May 2016.
  • Jackson M & Mantsch HH. The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure. Critical Reviews in Biochemistry and Molecular Biology 1995; 30: 95-120.
  • Kong J & Yu S. Fourier Transform Infrared Spectroscopy Analysis of Protein Secondary Structures. Acta Biochimica et Biophysica Sinica 2007; 39: 549-559.
  • Lawson D. A Brief Introduction to Protein Crystallography.
  • Sarroukh R, Goormaghtigh E, Ruysschaert J-M, et al. ATR-FTIR: A “rejuvenated” tool to investigate amyloid proteins. Biochimica et Biophysica Acta 2013; 1828: 2328-2338.

About Specac Ltd

SpecacSpecac manufactures an extensive range of FTIR Accessory, IR Polarizer, and Pellet Press Products for Atomic and Molecular Spectroscopy.

These products include ATR Accessories, Specular Reflectance Accessories, Diffuse Reflectance Accessories, Liquid Transmission and Gas Transmission Cells, as well as Infrared and Terahertz Wire Grid Polarizers, Bench-Top Hydraulic Presses, KBr Pellet Presses, XRF Pellet Presses, Thin Film Making Kits, and Evacuable Pellet Dies.

For online optical spectroscopy or FTIR analysis, Specac offers a comprehensive range of NIR Process Cells suitable for liquid and gas/vapour analysis.

Last updated: Feb 21, 2023 at 4:17 PM


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