FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media
Introduction
With the progress in sequencing of the human genome, the disparity between the number of known sequences and the number of experimentally determined protein structures continues to increase. Consequently, there is a clear demand for development of techniques for rapid structural characterisation of the encoded proteins. X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy provide the complete three-dimensional structure of a protein and are by far the most powerful techniques available to structural biochemists. Although X-ray crystallography is an excellent technique for the determination of three-dimensional structure of proteins it has the following disadvantages: crystallographic studies require high-quality single crystals which are not available for many proteins such as most of the membrane proteins, and the structure of a protein in a crystal may not always relate to its structure in solution. X-ray diffraction data presents a static picture of protein structure which does not represent the protein conformation with its dynamic nature in biological systems. The slowness of the procedure is the other disadvantage of the technique. NMR spectroscopy has better flexibility to study protein structure in solution. However, the interpretation of NMR spectra of larger proteins is very complex, and the technique is presently limited to small proteins (30 kDa).
It is important to point out that no one technique in the present arsenal of protein structural methods is able to provide information on all aspects of protein structure. Therefore, a rational strategy is to employ a concerted approach in which the protein is examined using several structural techniques. Information obtained from different techniques can be cross-correlated to provide a more complete picture of the chemical and physical state and/or bioactivity of the protein under different conditions. One of the techniques which has recently become very popular for structural characterisation of proteins is Fourier transform infrared (FTIR) spectroscopy. Here, we discuss the application of infrared spectroscopy for structure and stability studies of proteins in aqueous and non-aqueous media.
Infrared spectroscopy is based on molecular vibrations. Chemical bonds undergo various forms of vibrations such as stretching, twisting and rotating. The energy of most molecular vibrations corresponds to that of the infrared region of electromagnetic spectrum. Many of the vibrations can be localised to specific bonds or groupings, such as the CO and O–H groups. This has led to the concept of characteristic group frequencies. Typical group frequencies of interest to biochemists include CO, –COOH, COO−, O–H and S–H. There are many vibrational modes that do not represent a single type of bond oscillation but are strongly coupled to neighbouring bonds. For example, the infrared spectrum of a protein is characterised by a set of absorption regions known as the amide modes. With developments in FTIR instrumentation it is now possible to obtain high quality spectra from dilute protein solutions in H2O 1, 2, 3, 4. The overlapping H2O absorption can be digitally subtracted from the spectrum of the protein solution. In addition, the broad infrared bands in the spectra of proteins can be analysed in detail using second-derivative and deconvolution procedures 1, 2, 3, 4. These procedures can be utilised to reveal the overlapping components within the broad amide bands. Difference spectroscopy has the advantage in providing highly detailed information on conformational changes in proteins [5].
The most important advantage of FTIR spectroscopy for biological studies is that spectra of almost any biological material can be obtained in a wide variety of environments. Spectra of a protein can be obtained in single crystals, in aqueous solution, organic solvents, detergents micelles, lipid membranes, etc. The chemical environment in which a peptide or protein exists influences its structure and stability. This has important implications for the formulation, storage and delivery mechanisms for protein therapeutics. There is increasing evidence indicating that the environment can be important in determining the secondary structure formed by an amino acid sequence 6, 7. Other advantages of the technique include the following: the amount of protein required is relatively small (10 μg); the size of the protein is not important; there is no light scattering or fluorescent effects; kinetic and time-resolved studies are possible; and inexpensive compared to the cost of X-ray diffraction, NMR, ESR and CD spectroscopic equipments.
Section snippets
Instrumentation
Early studies of proteins using infrared spectroscopy was hindered by the low sensitivity of infrared spectrometers and absorption of liquid water over much of the infrared spectrum. With developments in FTIR instrumentation these problems have been solved. There are several companies that manufacture FTIR spectrometers and provide softwares for spectral analysis. A broad range of FTIR accessories are also available to overcome the problems of studying biological samples. For example, coupling
Data processing techniques
The amide I band of proteins consists of many overlapping component bands that represent different structural elements such as α-helices, β-sheets, turns and non-ordered or irregular structures. The width of the contributing component bands is usually greater than the separation between the maxima of adjacent peaks. As a consequence, the individual component bands cannot be resolved in the experimental spectra. The Fourier deconvolution procedure, sometimes referred to as `resolution
Investigation of protein structure and conformational changes
FTIR spectroscopy has been successfully used to investigate conformational changes in many different soluble proteins in aqueous solution (both H2O and ). Here, we present FTIR spectra of three proteins: Fig. 2 shows the FTIR spectra of a predominantly β-sheet containing water soluble protein in H2O and ; FTIR spectrum of a predominantly α-helical membrane protein in H2O is presented in Fig. 3; and finally Fig. 4 shows the spectra of a predominantly α-helical water-soluble protein in
FTIR spectroscopy of proteins in organic solvents and membranes
Often structural studies are carried out on proteins far removed from their natural environment. This can be for a deliberate purpose (for example, to monitor how the structure of a protein is influenced by its environment, and if this influences its function) or because one has no choice (since no suitable technique can be used for the structural analysis in the biologically relevant media). As an example of the latter situation, structure of membrane proteins and peptides are often
Structural characterisation of immobilised proteins
Immobilization of proteins and peptides using different procedures is becoming increasingly important for a number of medical and biotechnological applications. However, there is a distinct lack of techniques that can be used to characterise the conformation of such immobilised proteins. FTIR spectroscopy is one of the few techniques that can provide information in this area. The secondary structure of a chemically cross-linked protein can be readily characterised using FTIR spectroscopy as
Conclusion and future prospects
FTIR spectroscopy is a versatile tool that can be used to obtain information on different aspects of protein structure in a wide range of environments. Methods for quantitative estimation of protein secondary structure using this technique are progressively improving. Combining FTIR spectroscopy with secondary structure prediction methods provides a more reliable structure determination than is possible by using these methods individually. Second-derivative, deconvolution and difference
Acknowledgements
We would like to thank British Council-Academic Link Programme.
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