This project is designed to characterize the structural thermodynamics of a DNA binding protein from the hyperthermophile Sulfolobus. The emphasis is on both stability and function. Proteins from hyperthermophiles are of interest in protein engineering and biotechnology since they are designed for both folding and functioning at high temperature (via 80 to 110 degrees C). Little is known about the structures of hyperthermophile proteins, and even less is known about the thermodynamics of both their folding and function. The 7 kD chromatin proteins Sac7d and Sso7d are two of the few hyperthermophile proteins studied to date which unfold reversibly, permitting detailed thermodynamic studies. They also provide a simple model system for studies of the thermodynamics of non-specific DNA-binding using proteins that are designed for that purpose. Current ideas on the thermodynamics of nonspecific protein-DNA interactions are largely based on studies of sequence-specific proteins binding to non-consensus sequences. The first determination of the free energy of stabilization of a hyperthermophile protein was performed in the initial phase of this project using recombinant Sac7d. This work will now be extended to the native protein along with an extensive study of the folding thermodynamics with site-directed mutagenesis. Of particular interest will be a characterization and thermodynamic study of a post-translational modification, possibly lysine methylation, which increases the Sac7d stability. The importance of this modification, surface ionic interactions, ion binding, and core packing will be investigated by a combination of site-directed mutagenesis, NMR, chemical denaturation, and scanning and titration calorimetry. Sac7d and Sso7d bind double-stranded DNA non-specifically as monomers. This is an especially simple system for detailed structural thermodynamic studies of individual interactions and the thermodynamics of nonspecific binding. The importance of both ionic and nonionic interactions in DNA binding will be investigated by scanning and titration calorimetry, binding measurements using fluorescence and circular dichroism, and site-specific mutagenesis. Information gained from this project will contribute to our ability to design and control protein stability and protein-DNA interactions with potential applications in medicine and biotechnology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Wehrle, Janna P
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Southern Illinois University Carbondale
Schools of Medicine
United States
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Shriver, John W; Edmondson, Stephen P (2009) Defining the stability of multimeric proteins. Methods Mol Biol 490:57-82
Edmondson, Stephen P; Turri, Jacquelyn; Smith, Kelley et al. (2009) Structure, stability, and flexibility of ribosomal protein L14e from Sulfolobus solfataricus. Biochemistry 48:5553-62
Shriver, John W; Edmondson, Stephen P (2009) Ligand-binding interactions and stability. Methods Mol Biol 490:135-64
Clark, Andrew T; Smith, Kelley; Muhandiram, Ranjith et al. (2007) Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d. J Mol Biol 372:992-1008