Analytical Methods for Assessing the Effects of Site-Directed Mutagenesis on Protein-Cofactor and Protein-Protein Functional Relationships.

To completely understand the role of an amino acid residue that is targeted for site-directed mutagenesis a thorough analysis of the impact that the mutation has on the function of the protein is required. General methods for performing site-directed mutagenesis and expressing the recombinant protein variant are described. Protein-cofactor interactions are important because cofactors are often directly involved in facilitating catalysis by enzymes and in electron transfer by redox proteins. Many cofactors also have characteristic spectroscopic properties. As such, general methods are described to analyze the spectroscopic, redox and catalytic properties of protein-bound cofactors. Methods for assessing the effects of a mutation on protein-protein interactions are also described. Lastly, methods for assessing the overall structural integrity of the protein are described, as this is important to ensure that the mutation has not caused a global disruption of protein structure, rather than a specific effect on function.