Proper localization of proteins is crucial to all cells. The signal recognition particle (SRP) and its receptor (SR) constitute the major cellular machinery that delivers newly synthesized proteins to the eukaryotic endoplasmic reticulum membrane, or the bacterial plasma membrane. This process is regulated by two homologous GTPases in the SRP and SR that directly interact with one another, making it particularly exciting for mechanistic investigations. Though past work has defined the components of the targeting pathway, the molecular mechanism of this process remains unclear. Our general goal is to decipher, at a biochemical and biophysical level, the intricate inner workings of this universally conserved targeting machine. Our specific goal is to understand the mechanism by which the SRP and SR GTPases use their cycles of GTP binding and hydrolysis to provide spatial and temporal coordination of the protein targeting reaction. To this end, three specific aims are envisioned: (1) We will define and characterize the dynamics and conformational intermediates during SRP-SR complex formation and their reciprocal GTPase activation;(2) We will define whether, when and how other components of the protein targeting pathway - the ribosome, the signal sequence, and the membrane translocation channel - modulate the conformational changes of SRP and SR during their binding and activation cycle;(3) We will use the array of mutant GTPases and GTP analogues to perturb specific conformational steps in the GTPase cycle of SRP and SR, and test how these perturbations affect the recognition, delivery and unloading of cargo protein during the protein targeting reaction. These experiments will allow us to define the precise role of each GTP binding and hydrolysis event in providing the driving force or improving the fidelity of the protein targeting reaction. Ultimately, these studies will not only advance our understanding of the process of protein localization within the cell, but also provide new insights into the general principles of molecular recognition and regulation at a very fundamental level. The proposed research is of a most basic nature, and will contribute profoundly to our general understanding of physiology and pathology of all living cells at the molecular level.