An emerging lesson of modern biology is that living organisms cannot be understood as the sum of their component molecules but involve complex informational networks that behave as integrated systems. A second emerging lesson is that the biomolecular components themselves are not constant but change in structure and function during evolution. This project's research focuses on the functional evolution of important components of biomolecular networks, namely proteins that regulate gene expression by binding to certain patterns of nucleotide bases on double-stranded DNA. In a functionally diverse family of microbial DNA-binding proteins called Cro, analysis of databases suggested an "evolutionary code", whereby the preferences of the protein for single bases in target DNA appeared to evolve through simple, single amino-acid changes in the protein. The project involves testing the limits of this simple code by parallel application of site-directed mutagenesis, binding affinity measurements, binding site selection, and structural biology to diverse Cro proteins. These studies also include assessment of the contribution of more complex and subtle factors to functional evolution, such as evolutionary changes in protein structure. The educational portion of the project emphasizes opportunities for undergraduate research as a critical element in the development of a new generation of scientists, and incorporation of computer lab facilities in graduate-level teaching to facilitate a transition between classroom study and independent research. Broader impacts include development of the infrastructure for research and education through enhancement and use of computer lab facilities, increased opportunities for research experiences for undergraduates and students from underrepresented groups.