Abstract
Previous work in the laboratory of my research advisor, Dr. Wesley Stites, has investigated the core packing of the protein staphylococcal nuclease. The core of a protein is critical in determining a protein's structure and stability. The hydrophobicity of the core has long been thought to be the principal driving force for folding, but recent work in the Stites lab has shown that optimization of van der Waals contacts and minimization of cavities, in our shorthand term, packing, is at least as energetically important. We are building upon this information in our attempt to better pack the protein core. If we can do this, we predict that the improvement in packing will make the protein more stable overall. This project takes a closer look at the thermodynamically unfavorable left-handed alpha helix region in the core of staphylococcal nuclease. It has already been shown that there are angle strains on some of the residue; that can be repaired by replacing the leucine at residue 38 with glycine. However, this created empty space within the core that greatly destabilized the protein. Our hypothesis was that by filling this space with larger amino acids at nearby locations, we would be able to correct this problem. Several mutations were made at residues 38, 39, and 125. The residues are all within close contact with each other and in the vicinity of the left-handed alpha helix. The following procedures were used: Kunkel DNA mutagenesis, transformation and preparation of M13 single stranded DNA, transfer of nuclease mutant gene from M13 to plasmid, Laemmli discontinuous protein SDS-Page gel, protein preparation and purification, and fluorometric titration. The hypothesis that relieving angle strain near the lefthanded alpha helix with a glycine, and then filling the space caused by that mutation with larger amino acids near it would increase the protein's stability was confirmed. However, new packing problems were generated so most mutations resulted in an overall decrease in stability.
Recommended Citation
Danforth, Rebecca L.
(2004)
"The Effects of Multiple Mutations in the Hydrophobic Core Upon the Stability of Staphylococcal Nuclease,"
Inquiry: The University of Arkansas Undergraduate Research Journal: Vol. 5, Article 11.
Available at:
https://scholarworks.uark.edu/inquiry/vol5/iss1/11
