Theme 2 will examine the emergent complexity of metalloproteins in microbial ancestors. We will develop new computational methods for linking protein structure to the evolution of function, particularly to redox functionality of metal binding proteins. Methods for pairwise functional comparisons of metal-binding folds will be systematically constructed used data from the Protein Data Bank (PDB). We will then use available evolutionary tree-building techniques, augmented with newly developed approaches, to evaluate relative time of appearance of all folds in our set.

Internal tree nodes, the imputed potential ancestral folds of extant proteins, will be synthesized in the lab to verify the tree topology. The composition of ancestral folds in terms of Theme 1 motifs, as well as their function and structure will also be assessed. Finally, we will design computational methods for mapping mineral/metal/trace-element availability to our evolutionary tree. In this manner we will be able to identify the oldest metal-binding protein structures and to time the early evolution of metal binding folds. The machine-learning component of this final task will also allow us to identify trace-element/metal signatures indicative of the various stages of development of life.