Energetics of Protein Folding and DNA Interactions

Our laboratory is devoted to characterizing the energetics of protein folding and ligand binding with a special emphasis on DNA interactions that are essential in many normal cellular functions such as gene regulation, expression, recombination, and chromatin organization. We take advantage of the high stability of DNA binding proteins from hyperthermophiles such as Sulfolobus to obtain unusually precise and accurate data that are difficult to obtain on less stable mesophile proteins. Sulfolobus is an Archaea which grows in numerous thermal hot springs around the world (e.g. Yellowstone National Park). Shown below is the Sulfolobus acidocaldarius protein Sac7d (blue) bound to an 8 base pair segment of DNA (white). Sac7d and the homologous Sso7d from S. solfataricus (Abstract) are excellent examples of a chromodomain, a fold common in eukaryotic nuclear proteins that is also found in the DNA-binding domain of HIV-1 integrase. The protein binds to DNA via mechanisms similar to those observed in proteins with direct biomedical relevance. The structure of the complex shown here was determined by X-ray crystallography by Andy Wang (Abstract). The structure of the protein free in solution has been determined by us using NMR and is essentially identical to that when bound to DNA (Abstract). Note that binding results in intercalation of valine and methionine side chains (green) into the DNA and leads to a significant kink in the DNA with one of the largest roll angles observed at a single basepair step. Similar interactions occur in a number of important protein-DNA complexes. The small size of Sac7d and its high stability permit a detailed characterization of how this type of interaction affects the binding affinity (Abstract).

The long term goal of our 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. Sac7d is an excellent hyperthermophile protein for studies of protein stability (Abstract) and flexibility (Abstract). 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 our work we take advantage of the high thermal stability of not only Sac7d, but also other DNA binding proteins from Sulfolobus to quantitatively describe the importance of various interactions in folding and the energetics of binding and distorting DNA in protein-DNA complexes. In combination with structural studies using NMR we also determine the stability of the proteins and their complexes with differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) (Abstract).

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. The 7 kD chromatin proteins Sac7d and Sso7d have proven to be ideal hyperthermophile proteins for studies of stability (Abstract). Given a high density of ion pairs and our ability to define the stabilities of these proteins over a wide range of conditions (Abstract), they are ideal for studies of the importance and behavior of ionic interactions in hyperthermophile proteins.

We have also recently begun studying two other DNA binding proteins from Sulfolobus: Sso10a and Sso10b. These are two very different proteins that both exist as dimers in solution. Sso10b is sometimes referred to as Alba and may play a role in DNA compaction as well as RNA metabolism (see, for example, the work of Aravind et al.). Two views of the NMR structure of Sso10b2 are shown here. The structure is more completely defined in a recent publication (Abstract).

Sso10a is an interesting protein that most likely serves as a transcription factor in Sulflobus. It is a dimer of winged helices with an interesting dimer interface composed of an extended and very exposed antiparallel coiled coil (Abstract). The coiled coil contains a buried salt bridge that may play a role in defining the register and stability of the dimer. This structure is the first to be determined of a conserved family of DNA-binding proteins referred to as COG3432 (Abstract).

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.

url: http://chemistry.uah.edu/shriver.htm

The University of Alabama in Huntsville, Huntsville, AL 35899

site updated: 04 January 2006(jws)