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There have been many approaches to solving problems associated with protein solubility. This article describes the application of directed evolution to improving the solubility of the C-terminal metal-binding domain of aminopeptidase P from Escherichia coli. During the course of experiments, the domain boundary and sequence were allowed to vary. It was found that extending the domain boundary resulted in aggregation with little improvement in solubility, whereas two changes to the sequence of the domain resulted in dramatic improvements in solubility. These latter changes occurred in the active site and abolished the ability of the protein to bind metals and hence catalyze its physiological reaction. The evidence presented here has led to the proposal that metals bind to the intact protein after it has folded and that the N-terminal domain is necessary to stabilize the structure of the protein so that it is capable of binding metals. The acid residues responsible for binding metals tend to repel one another − in the absence of the N-terminal domain, the C-terminal domain does not fold properly and forms inclusion bodies. Evolution of the C-terminal domain has removed the destabilizing effects of the metal ligands, but in so doing it has reduced the capacity of the domain to bind metals. In this case, directed evolution has identified active site residues that destabilize the domain structure. [ABSTRACT FROM AUTHOR]

Directed evolution is a useful tool in the study of enzymes. It is used in this study to investigate the means by which resistance to the antibiotic trimethoprim develops in dihyrofolate reductase from Escherichia coli. Mutants with clinical levels of resistance were obtained after only three generations. After four generations of directed evolution, several mutants were characterized, along with some point mutants made to investigate amino acid changes of interest. Several mutations were found to grant resistance to trimethoprim, both by reducing the binding affinity of the enzyme for the drug, and by increasing the activity of the enzyme. [ABSTRACT FROM AUTHOR]