23 Jun 2016

Using FTIR spectroscopy to determine protein secondary structure

Proteins are the building blocks of life. They play a very important role in the function and structure of cells and are involved in the body’s transport, communication and metabolism.

Only 20 amino acids are used to form proteins, however their potential structures, sequences and combinations are almost infinite. FTIR spectrometer accessories such as the Pearl transmission accessory, are vital tools for sample characterization and measurement.

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All proteins fulfill a certain purpose, often depending on their three dimensional structure. Therefore, establishing the definition of protein 3D structure is important for learning how they work and can provide information on how proteins interact with other molecules, function as enzymes and undergo conformational changes. The data can be useful for drug design and for the manufacture of industrial enzymes.

If you want more information on the Pearl liquid FTIR transmission cell, get in touch for a price and product demonstration.

High-resolution approaches

The best method for understanding protein structure is X-ray crystallography. But it can be limited by the fact that proteins often don’t form crystals or form crystals that don’t resemble the natural or native state of the protein.

Another high-resolution method is nuclear magnetic resonance (NMR) spectroscopy, which can characterise protein structure when in liquids. Unfortunately, this method is only practical for studying smaller proteins, measuring 15 to 25kDa. And both of the aforementioned techniques require specific sample preparation steps, as well as specialised skills. Therefore, they can take up considerable amounts of time.

A good alternative for determining secondary protein structure is Fourier transform infrared (FTIR) spectroscopy. This method can acquire spectra in many environments and across various different protein sizes. It is also quicker and requires less sample preparation than most other techniques.

Insights into Structure

In typical FTIR, infrared light is shone onto a sample and measurements are made of how specific wavelengths of IR light are reflected or absorbed. Characteristic absorption bands occur from various structural areas 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 gives valuable insights regarding the secondary structure, as both of these forms of bonding are influenced by the protein’s secondary structural content. Amide I bands, especially, are sensitive to this secondary structure.

FTIR spectroscopy can also allow information on proteins’ structural dynamics to be obtained. Protein activity can be influenced by the structural dynamics of a specific conformation and these dynamics are important for the protein performing its function. Information on structural dynamics is found by evaluating the rate of exchange of hydrogen isotopes (e.g. deuterium) in water with hydrogen atoms in the protein. This displays 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 is also valuable for the study of protein stability. This includes the folding and unfolding processes under various thermal and chemical conditions and the protein aggregation process, which has been oft studied in Huntington's, Alzheimer's, Parkinson's and other amyloid diseases. Furthermore, FTIR spectroscopy discovers data on protein side-chains – the sequence of R groups that are part of and distinguish the amino acids present in a polypeptide chain.

Although FTIR spectroscopy can’t give the same high-resolution data provided by other methods such as NMR and crystallography, it offers a fast and efficient screening of proteins in their native state for a fraction of the cost of the other methods.

A Vital Accessory

The main benefit of FTIR over other techniques is its convenience. This is taken further by the Specac Pearl spectrometer accessory, which is compatible with almost any spectrometer on the commercial market. The special liquid transmission Oyster Cell (horizontal liquid sample cell) design allows the easy addition of liquid samples to the accessory, without preparation, by injection or spotting. Cleaning is equally easy.

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Specac's Pearl can also be used to study viscous liquids that could not be handled by traditional analysis accessories. And finally, the Pearl gives accurate and repeatable pathlengths and offers a wedging option which prevents fringing, as well as interchangeable windows to accommodate various pathlengths.

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References

  1. Gallagher W. FTIR Analysis of Protein Structure. Available at: http://www.chem.uwec.edu/Chem455_S05/Pages/Manuals/FTIR_of_proteins.pdf. Accessed: May 2016.
  2. 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.
  3. Kong J & Yu S. Fourier Transform Infrared Spectroscopy Analysis of Protein Secondary Structures. Acta Biochimica et Biophysica Sinica 2007; 39: 549-559.
  4. Lawson D. A Brief Introduction to Protein Crystallography. Available at: https://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htm. Accessed: May 2016.
  5. 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.