Physical characterization of proteins: FTIR, ATR-FTIR, DSC and THz imaging
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In recent years the number of tools available for the analysis of biological properties of proteins has advanced significantly. A wide facet of a protein's intrinsic physical and biological properties such as protein secondary structure and melting/freezing points can be examined using physical techniques. These techniques have broad applications in both discovering novel properties of proteins or serve as pertinent methods to provide additional information on protein properties. Here, three techniques to examine protein properties are undertaken. FTIR is a well-described technique to identify unknown samples due to unique absorption patterns. We show an alternate application of the method. In order to determine the secondary structure present in denatured Hen Egg White Lysozyme (HEWL), Fourier-Transform Infrared Spectroscopy (FTIR/ATR-FTIR) was employed to compare the levels of protein secondary structures (i.e. α-helices, β-sheets and random coils) in both native and denatured HEWL protein. It was found that secondary structure was indeed still present in HEWL denatured with 6M Guanidine hydrochloride (GdnCl). This served as important confirmation of a finding that the protein dynamical transition required minimal secondary structure but not tertiary structure. Next Differential Scanning Calorimetry (DSC) was conducted to investigate whether the abrupt change of transmission at various frequencies at 283K in a plot of refractive index as a function of temperature represented the melting and freezing point of the water changed due to the presence of the Cytochrome C protein. Based on the results, we were able to effectively rule out that this represented the melting or freezing point possibility. Lastly, results from terahertz apertureless near-field scanning optical microscopy (THz-ANSOM) were analyzed in order to decipher important biological properties of lysozyme crystals. THz-ANSOM has the advantage of being harmless to biological molecules and that most samples are transparent to THz lights and that biological molecules may have a unique spectral fingerprinting pattern in the THz range. Moreover, THz-ANSOM techniques provide a method to examine the spectral responses for Small samples. Therefore it is clear that these techniques are not only useful in uncovering novel properties of biological molecules but can also be applied to the fine-tuning and support of findings from larger scale experiments.