STRUCTURAL THEMODYNAMICS OF A HYPERTHERMOPHILE

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Hyperthermophile proteins are of special interest in structural biology since they are designed to function at the upper temperature limit of life (viz. 80 to 110 degrees C). The long term goal of this work is to utilize the unique attributes of hyperthermophile proteins to increase our understanding of protein energetics and enhance our capabilities in protein engineering and biotechnology. These proteins are important not only for providing clues on how to control stability in a rational manner, but also for expanding the experimental range of conditions available for investigating protein processes such as folding and binding. In this work we propose to take advantage of the high thermal and acid stability of two hyperthermophile proteins to quantitatively describe the importance of ionic interactions in folding and the energetic cost of distorting DNA in protein-DNA binding. The structures of a number of hyperthermophile proteins are now available, and this data has been utilized to propose rational explanations for enhanced stability and function in these proteins at high temperature. The most common explanation is a greater number of ion pairs, which are expected to be increasingly important at high temperature. There have been few in-depth thermodynamic studies of the relative importance of the proposed ion pair interactions in these proteins and no experimental demonstration of the predicted temperature dependence. The 7 kD chromatin proteins Sac7d and Sso7d have proven to be ideal hyperthermophile proteins for studies of stability. Given a high density of ion pairs and our ability to define the stabilities of these proteins over a wide range of conditions, they are ideal for studies of the importance and behavior of ionic interactions in hyperthermophile proteins. Sac7d and Sso7d also provide relatively simple model systems for studies of the energetics of protein-DNA binding since they induce one of the largest kinks in DNA observed for any protein. The high thermal stability will be utilized here to define the energetics of DNA binding and bending over a wide temperature range to reliably separate the effects of folding, binding and bending. These interactions will be investigated by a combination of scanning and titration calorimetry, NMR, site-directed mutagenesis, circular dichroism, and small-angle X- ray scattering. Information gained from this project will contribute to our ability to design and control protein stability and protein-DNA interactions in a rational manner with potential applications in medicine and biotechnology.