DESCRIPTION Abstract: The success of crystallography in determining atomic resolution structures of thousands of diverse proteins has revolutionized biology. Membrane proteins, a large and medically important class, have not kept pace with the advances in methodology that have made this revolution possible. The reason for this is that, unlike soluble proteins, membrane proteins must enter into the lipid bilayer during their biogenesis. This additional level of complexity has prevented membrane proteins from being obtained at levels similar to soluble proteins. For structural biology to be successful, a membrane protein must be overexpressed. The problem is that with current technology this has been impossible for the majority of membrane proteins; therefore, few structures are determined and each is extremely expensive. This proposal aims to solve this problem by proving the following hypothesis: The limiting factor in membrane protein overexpression is their need to be targeted and inserted into the membrane. Membrane proteins are recognized by N-terminal signals that must be accurately read to appropriately deliver the protein for insertion into the membrane via the translocation machinery. Our goal will be to develop a universal signal that will allow us to overcome this intrinsic rate- limiting step. We will accomplish this by systematically testing every aspect of membrane protein targeting. With this comprehensive approach, we will derive biological insight; moreover, we will dramatically improve the number of membrane protein targets available for crystallization. The effect will be to improve the success rate of crystallization trials dramatically reducing the cost of solving a membrane protein structure. The hope is to open up a new revolution in membrane structure determination that will allow us to address a wealth of important medical problems. My laboratory is uniquely positioned to lead this new revolution. ) Public Health Relevance: Atomic resolution structures prov