An estimated 20-30% of all open reading frames in the eukaryotic cell encode a membrane protein and an estimated 60% of all drugs bind to a membrane protein. Not surprisingly, statistical analysis of the current drugs indicate that hydrophobicity is one of 8 properties that is common in a drug target. However, the number of high-resolution structures of membrane proteins is relatively small and the number of structures of hydrophobic molecules bound to membrane proteins is even smaller. This proposal has 2 disparate projects whose only commonality is that the proteins under investigation are membrane proteins and involved in critical functions in the cell. The first project entails the yeast mitochondrial ATP synthase of which we have solved the first near atomic model of the entire monomeric enzyme and we did this, with and without inhibitors bound to the membrane embedded, Fo domain. We propose to expand these studies to provide structures of the ATP synthase inhibited or trapped in various reaction intermediates. Our approach is unique in that we have linked 2 subunits of the ATP synthase and this allows us to capture intermediates with the rotor in a twisted state. As such, our approach allows for the capture of reaction intermediates. The use of inhibitors will serve to trap intermediates and identify their mode of binding. The second project is studies on yeast Yhc3p, a homologue of human Cln3p, which is in humans, is defective in the juvenile neurodegenerative disease, Batten. While, Cln3 was reported in 1995 as the gene defective for the juvenile form of Batten disease, the function of this protein is still unknown. Based on homology and other data, we hypothesize that Cln3p is a small molecule transporter whose function is tied to the formation of oxygen radicals. We have evidence that the yeast gene, YHC3, is tightly regulated and believe that by identifying the regulation, we will understand its importance in the cell. Lastly, we have developed an over-expression system and purification scheme for Yhc3p, which we will use to study the function and start crystallization trials for x-ray diffraction studies. While disparate, these projects fall well within our expertise and will add to the base necessary for the understanding of membrane proteins in health, disease, and as targets for drug discovery. !
This study has two disparate goals: 1) we will advance our understanding of the mechanism of ATP synthase and coupling of ion translocation to the synthesis of ATP. 2) we will advance our understanding the function and structure of the protein defective in the neurodegenerative disease, Batten disease. The results herein will lead to advances in the treatment of diseases and adverse health conditions.
