Terahertz spectrometry applied to proteins
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Electromagnetic radiation from the radio waves used in nuclear magnetic resonance spectroscopy through to X-rays used in crystallography have provided a wealth of knowledge about the structure, function, and dynamics of protein molecules. Terahertz waves, the topic of this thesis, are lower in frequency than radiation from the infrared, not to the frequencies of individual bond vibrations, but to the frequency range where slower longer range protein librations (low frequency vibrations) are expected to occur. The role of low frequency protein dynamics remains controversial, with some arguing that these motions are crucial for enzyme and protein function. Terahertz spectroscopy may provide key evidence to contribute to this interdisciplinary debate. In this thesis, terahertz (THz) spectroscopy has been applied in studying a number of proteins experimentally. In the first results chapter, the effect of protein concentration and ionic strength in the 0.1-2.5 THz region was investigated using Terahertz time domain spectroscopy. The results confirm the presence of terahertz excess for a number of proteins, which results from the increased absorption of THz waves when protein is introduced into the system. THz spectroscopy was then used to detect the difference between a folded protein, myoglobin, and folding intermediates, including the molten globule form, apomyoglobin. The results collected using THz spectroscopy were unable to differentiate between the folded and molten globule states. A further study was susceptible to the formation of higher order protein complexes and explored structures formed using PduA*. These experiments were primarily biochemical in nature with showing that PduA* assembles into nanotubes of 20nm diameter in vitro. The final results chapter explores the sub-THz circular dichroism signal from a vector network analyser driven by quasi-optical circuits. Wherever possible, the THz experiments were benchmarked using established analytical techniques.
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