Small angle X-ray scattering studies on proteins under extreme conditions

Abstract

Proteins are a class of biologically relevant macromolecules that are ubiquitous in all living organisms and fulfil specific functions in these. Essential for most proteins is the coupling between their three-dimensional structure and their biological function. Different external extreme conditions like heat or pressure might lead to the loss of the protein's complex structure, so-called unfolding, and thus of its activity. To protect proteins from these extreme conditions different mechanisms have evolved in organisms to counteract these perturbations. A detailed knowledge of those factors that govern protein stability and how these are affected by external perturbations and modulated by different cosolvents might give a better understanding of how life has evolved under such conditions and what conditions are still tolerable for life to exist. In order to study specific contributions to the protein stability in a controlled way, com binations of different extreme conditions can be employed in an experiment to induce unfolding. In the framework of this thesis, proteins under extreme conditions were studied by small angle X-ray scattering (SAXS). This technique is well suited to determine the size and shape of proteins and thus to investigate their unfolding due to perturbations. Furthermore, using concentrated solutions it can be used to explore the interactions between proteins. This gives the possibility to learn about the factors that govern interactions in dense protein solutions as they are present in the cell's interior. SAXS measurement were performed on Different types of proteins at various extreme conditions to access the influences of the specific properties of the local and global structure on the protein folding. Therefor, SAXS data were obtained for various mutants of staphylococcal nuclease (SNase), a small globular protein, at high pressures and different pH values. This approach allowed to detect the influence of the charge properties of a single amino acid on the global folding state. Moreover, the structure of highly destablized SNase mutant was investigated as a function of solution pH. In addition, specific features of the linear, nonglobular ankyrin repeat domain were determined as a function of pressure and urea concentration. SAXS studies on highly concentrated solutions of the protein lysozyme were conducted at high pressure conditions. These measurements allowed it to determine the pressure-dependent protein-protein interaction potential. For this interaction potential a nonlinear pressure dependence was observed which can be interpreted by the collapse of the second hydration shell of water at 2 kbar. In addition, the influence of small biologically relevant molecules known to affect the protein stability especially under high hydrostatic pressures were studied. The effect of the osmolyte TMAO, which present in deep sea organisms to compensate the effects of high pressure and urea, as well as the influence of urea, glycerol, and mixtures of TMAO/urea and TMAO/glycerol was analyzed. This SAXS measurements on proteins with TMAO added revealed a different pressure dependence of the interaction potential than for pure buffer. In contrast no effect was observed for urea and only a slight one for glycerol. For a 1:2 TMAO:urea mixture as it is present in deep sea organisms, a cancelling of the effects of the two substances was found.