PROJECT SUMMARY/ ABSTRACT Hedgehog protein is unique by being covalently modified by cholesterol. This is critical to its signaling role in embryonic development, and also in regulating adult stem cells. Malfunction of hedgehog signaling leads to severe birth defects and is involved in many cancers. Cholesterylation is essential to Hedgehog function and is catalyzed by the C-terminal domain of the Hedgehog precursor protein. Many of the deleterious effects of mutations in cholesterol biosynthesis are thought to involve Hedgehog signaling. The sterol binding region of the catalytic domain is poorly understood because it is intrinsically too disordered to be amenable to crystallography or protein NMR. The overall goal of this proposal to provide information about the sterol-binding domain through the use of modified sterols. A library of 23 sterols bearing a methyl group at each position of the sterol nucleus will be assembled by organic synthesis. The methyl groups will be put in place by methylenation of the corresponding ketones, followed by reduction. Both stereoisomers will be obtained at each secondary position. The angular methyl groups will be introduced by literature methods. Where precedence does not exist (positions 8 and 9), attempts will be made to apply methods for C5-methylation. This library will be tested by a collaborator in a FRET-based Hedgehog processing assay. The results will establish which sites of the sterol contact the binding site of the protein. The synthetic strategy allows adding steric bulk (methyl to ethyl) to better define the binding region. Mutagenesis studies by the same collaborator which regain function with a blocked sterol will determine which amino acids contact which regions of the sterol molecule. Another library of 23 sterols, this time bearing fluorine atoms at each position, will be synthesized using largely the same precursors that were used to make the methyl sterol library. This library will be used for 19F-NMR based studies to map the binding site of the protein. Covalent inhibitors of the processing reaction based on this library will also be synthesized and studied. The NMR studies will be carried by a collaborating structural biologist. Hedgehog protein modified by the sterol analogs may be useful to study downstream signaling interactions. The two sterol libraries may be useful in the study of other protein-sterol interactions.