Experimental analysis of helical protein stability

My dissertation research focuses on evaluating the determinants of protein stability. Developing a rational method to increase protein stability could be of practical utility for developing new enzyme based technologies and improving therapeutic proteins. Investigating the structural motifs of proteins from extremophiles, members of the archael kingdom that survive in harsh environments, has provided clues to the basis of protein stability. These include: increased ion pairs and ion pair networks, improved helix capping, decreased flexibility, tighter hydrophobic packing of the protein core, additional aromatic-aromatic interactions and increased hydrophilicity of the surface. Metal ligation is an additional approach to enhance protein stability. This method involves the introduction of metal binding amino acids, which upon chelating, stabilize the protein by rigidifying region(s) of a protein that may be susceptible to degradation or fraying.; Using a cloned single box (rHMGB1b) of the DNA binding protein HMGB1b, I explored the role of surface ion pairs and metal ligation stabilizing the native state of proteins. High Mobility Group (HMG) proteins are an abundant class of DNA binding proteins located in the nucleus, active in chromatin structuring and regulation of gene expression. The design principle used site directed mutagenesis to introduce sets of ion pairs and His-His ligand binding sites that replace neutral side chains in the binding and non-DNA binding surfaces of the protein. We replaced amino acids that make up the C and N-termini of two converging alpha-helices separated by a flexible loop. The loops were rigidified with "clamps" via ion pairs or metal binding His-His variants locking the molecule in place. The evaluation of metal binding His-His motifs also relates to a study involving the alpha helical nucleation and stabilization of small peptides. In this system, histidine residues spaced 3 residues apart are placed at the ends of an alanine based peptide. Upon binding of the histidine residues to a respective metal (NiII), the helicity of the peptide is increased by 40.6%, with an increase in stability of -1.1673 Kcal·mol-1. These experiments provided the "seed" data to evaluate the rHMGB1b. Varying the numbers of groups involved and the geometry of the ion pair(s) and His-His binding motifs(s) shows that stabilizing this protein was feasible. The most stabilizing mutant is a metal binding His-His variant that increases the unfolding temperature by 6.4°C. More importantly the relationship of protein stability and function was investigated. The stabilized His-His variant retained DNA binding affinity, indicating that an increase in rigidity (loss of flexibility) of a molecule does not compromise its function.