Design and application of sensitive detection methods for the study of biological systems

Molten globules are compact intermediates in the protein folding process which retain native- like secondary structure, but have a fluctuating ensemble of tertiary folds. Human alpha-lactalbumin is a Ca2+ binding protein which forms a molten globule at pH 2. Although much is known about the secondary structure of the alpha-lactalbumin molten globule, the tertiary structure has so far not been elucidated. Presented in Chapter 2 is a double electron-electron resonance study of alpha-lactalbumin. The C-helix was found to be fully folded in the molten globule, in agreement with previous studies. Furthermore, it is also shown that both the N- and C-termini are in close proximity to the C-helix, forming part of the hydrophobic core of the protein. Data collected in the presence of 8M urea show that the protein is unfolded under these conditions, and distinguishes the molten globule state from this. These data provide quantitative information about the tertiary structure of the alpha-lactalbumin molten globule, and may provide important insight into the protein folding process. Although a wide variety of chemical and biological systems are known to be sensitive to the strength of an applied magnetic field, no biological systems, such as the cryptochrome proteins believed to be the magnetoreceptors in migratory birds, have yet been shown to be sensitive to the field direction. Traditionally, the anisotropic response of such systems to the magnetic field has been probed using photoselection or alignment with liquid crystals. The experimental sensitivity to anisotropy could, however, be improved by increasing the degree of molecular alignment, such as by crystallisation. Presented in Chapter 3 is a study of hen egg white lysozyme crystals doped with FMN, which together form a magnetically sensitive system. The crystals were studied using confocal microscopy, with the magnetic field effect being spatially resolved. The diffusion of FMN through the crystals was also studied, with the spatial variation of the magnetic field effect being explained with reference to the diffusion of FMN. Although no anisotropy of the magnetic field effect was observed in this system, the use of crystallisation as a method of controlling molecular orientation is potentially useful in systems which are expected to show anisotropy (such as cryptochrome). The behaviour of many chemical systems differs between the bulk solution and at the interface. These interfacial processes can be difficult to probe using conventional absorption spectroscopy due to the relatively low absorbance of species at the interface as compared to the bulk. Cavity ring down and cavity enhanced absorption spectroscopy are two techniques which increase the sensitivity of absorption measurements, and evanescent wave variants of both have been applied to interfacial studies. Presented in Chapter 4 is a polarisation sensitive, broad band, evanescent wave cavity enhanced absorption experiment, which allows the study of the spectra of species at the silica/solvent interface across the visible region. A variety of systems which show interesting inter- facial behaviour have been studied, including crystal violet at the silica/water interface, and with polyelectrolyte films. The data could be fitted to extract spectra, kinetics, and average orientations of three individual species. These studies demonstrate the power of the polarisation sensitive, broad band nature of this experiment in determining complex interfacial behaviour involving more than one species.