One of the most intriguing questions in evolutionary biology is how biochemical and structural complexity arise through small and incremental changes; however answering this question requires an explicit set of candidate residues and an experimental system in which to test them. This dissertation aims to understand how biochemical complexity evolves and assesses the structure-function relationship in the green fluorescent protein (GFP) protein family using an ancestral reconstruction approach. In the second chapter, I studied the evolution of biochemical complexity in Kaede-type red fluorescent proteins (FPs) from Faviina corals. An increase in biochemical complexity is represented by the emergence of red fluorescence because it necessitates the synthesis of a tri-cyclic chromophore from a precursor bi-cyclic chromophore through an additional autocatalytic reaction step. The autocatalytic reaction is fully enabled by as many as twelve historical mutations. Here, I showed that the red fluorescent chromophore evolved from an ancestral green chromophore by perturbing the ancestral protein stability at multiple levels of protein structure. Moreover, only three historical mutations are sufficient to initiate the selection-accessible evolutionary trajectory leading to emergence of red fluorescence. The third chapter investigates six mutations proximate to the chromophore in the Kaede-type FP that could have facilitated autocatalytic synthesis of the red chromophore by enlarging the chromophore-containing cavity and modifying its microenvironment. Two of these six mutations were found to strongly affect the protein’s stability and oligomeric tendency. Additionally, I showed that the dimeric least divergent Kaede-type FP, R1-2, evolved from the tetrameric green ancestor. Taken together the results of these studies indicate that the step-up in biochemical complexity in the Kaede-type FPs was achieved via disruption of the existing stable interactions at tertiary and quaternary protein structure levels. In the fourth chapter, I resurrected the common ancestor of all FPs cloned from the order Leptothecata (class Hydrozoa), which are characterized by the highest known homo-oligomeric diversity. I showed that the ancestor was a green monomeric FP with a large Stokes shift. The ancestral FP together with the extant Leptothecata FPs could server as a model system to study the evolution of function and homo-oligomerization, and the desirable photophysical characteristics would make this ancestral FP a useful bio-marker in bio-medical research. / text