The three projects discussed in this thesis are unified by the common goal of understanding and manipulating protein function from an energy landscape-based perspective.

First, I explore how the energy landscapes of ribonucleases HI have evolved over time by resurrecting and characterizing extinct ancestors to the modern-day homologs from E. coli and T. thermophilus. Our results suggest that thermostability is a finely tuned property, which has adapted along each evolutionary lineage of RNase H to accommodate diverse environments. The thermodynamic mechanisms by which these changes occur, however, are found to be highly variable.

Then, I describe the construction of an unfolded maltose-binding protein and its subsequent analysis using neutron scattering to probe pico-nanosecond dynamics on the protein's surface. We find that our model for the unfolded state is more dynamic than its folded state and, perhaps more surprisingly, also more dynamic than an intrinsically disordered protein, tau. This interesting result highlights the difference between proteins that have evolved to be disordered and the unfolded state of proteins that have a well-defined native state.

Finally, I design and characterize an enzymatic switch that responds allosterically to a novel effector. The design is based on the principle of mutually exclusive folding and involves fusing a ligand-binding protein with an enzyme to create a construct in which only one domain is folded at a time. Several of the constructed chimeras are inhibited by ligand, and the strengths and weaknesses of our design are discussed.