Project Summary/Abstract Although its significance for biology has been recognized for more than a century, the molecular mechanism of allostery remains the subject of intense research. In allostery a signal from one site in a protein is transmitted to a second, often distant, site to alter its function. The ensemble model provides a framework in which dynamic and/or structural changes can contribute to allosteric regulation even within a single protein. The challenge lies in deciphering which molecular processes are critical to signal transmission. In this work we will investigate the allosteric mechanism in the Group II Biotin Protein Ligase, E.coli BirA protein. We hypothesize that BirA allostery occurs through direct coupling of dynamic changes in multiple loops to formation of residue networks. We further hypothesize that this mechanism allowed for the evolution of allostery while preserving an essential enzymatic function in post-translational biotin addition. These hypotheses will be tested using integrated genetic screening, solution biophysical measurements, x-ray crystallography, and molecular dynamics simulations. The elucidation of how allostery works in BirA will improve our general understanding of allosteric mechanisms and may prove useful for developing drugs that selectively target specific microbial BPLs and for creating Biotin Protein Ligase-based technology tools.