With nearly 1,350 complete genome sequences available our understanding of biology at the molecular level has never been more complete. A consequence of these sequencing projects was the discovery of large functionally unannotated segments of each genome. The genes (and proteins they encode) found in these unannotated regions are considered "hypothetical proteins". Current estimates suggest between 12%-50% of the known gene sequences are functionally unannotated. Incomplete functional annotation of the various genomes significantly limits our understanding of biology. Pragmatically, identifying the functions of these proteins could lead to new therapeutics; making functional annotation of paramount importance.

This dissertation describes the development of new methods for protein functional annotation independent of homology transfer. The hypothesis is proteins with similar function have significantly similar active sites. Nuclear magnetic resonance ligand affinity screening was employed to identify and define protein active sites. The methods developed were tested on a series of functionally diverse, annotated proteins including, serum albumins ( H. sapiens, B. taurus), α and β amylases (B. licheniformis, A. oryzae, B. amyloliquefaciens H. vulgare, I. batatas), primase C-terminal domain (S. aureus), nuclease ( S. aureus) and the type three secretion system protein PrgI ( S. typhirium).

Functional annotation using protein active sites require a high-resolution three-dimensional structure of the protein. In addition to method development, this dissertation describes the NMR solution structure of Staphylococcus aureus primase carboxy-terminal domain (CTD). The primase CTD is essential for bacterial DNA replication and distinctly different from eukaryotes. With the rapid rise in antibiotic resistance, the primase CTD of S. aureus is an attractive antibiotic target. The methods used for functional annotation were used to screen S. aureus primase CTD to identify the compound acycloguanosine as a binding ligand to primase CTD.