PROJECT SUMMARY/ABSTRACT Sugars, in particular glucose, are bioenergetic molecules involved in a broad range of essential cellular processes. These metabolites, and their derivatives, are essential intermediates in glycolytic pathways leading to the production of ATP and are further used in the glycosylation of proteins and other macromolecules as part of the biosynthetic-secretory pathway. In humans, glucose is absorbed in the small intestine by enterocytes and delivered to the bloodstream. Blood glucose concentrations are tightly regulated by hormonal control and conserved reabsorption mechanisms in the kidneys. Secondary active transporters facilitate these absorption/reabsorption processes as well as the exclusive delivery of activated sugar molecules to the ER and Golgi. Disturbances in these functions are associated with numerous human disorders, such as type II diabetes. It is, therefore, a critical objective of biomedical research to understand the structural intricacies of these dynamic transporters. During my 15-year tenure as an independent investigator, my lab has produced a number of experimental firsts—the first crystal structure of a sodium glucose transporter, the first crystal structure of the Voltage Dependent Anion Channel, and the firsts to use these coordinates for their biochemical and biophysical characterization. The last five years have been extremely fruitful for my lab. We have complimented these original structures with new structures in distinct conformations and incorporated their biophysical characterization in effort to elucidate their transport mechanisms. These findings have direct implications to our collective understanding of associated diseases and aid in drug development. This has been made possible due to my long-standing R01 funded by NIGMS that is currently in its 14th year. Additionally, I was recently awarded a new R01 that aims to elucidate the structural basis of transport for Nucleotide Sugar Transporters. These sources of NIGMS funding have allowed my lab to answer fundamental questions regarding sugar transport and cellular processing. This current proposal embodies the spirit of the MIRA funding scheme by allowing me to tackle bigger questions that are frequently referred to as `Higher-Risk' or `Ambitious Science', but that are the results every scientist truly dreams of acquiring. Elucidating the structure-function relationship of membrane transporters is particularly risky and requires a long- term commitment and flexibility to explore different directions of research and methodologies and developing new approaches. The stability and flexibility incorporated into the MIRA allows us to do exactly that: try new exploratory research, which will not only determine structures of human transporters that are direct pharmaceutical targets, but also delineate their mechanism of transport that will clearly be applicable to many other transporters in general.