Biophysical Studies of Bifunctional Peptidylglycine alpha-Amidating Monooxygenase.

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Dr. Merkler from the University of South Florida (USF), Dr. Dempsey from Boston University, and Dr. Richards from the Foundation for Molecular Evolution are investigating peptidylglycine α-monooxygenase (PAM), the enzyme responsible for making a diverse family of neuropeptides in humans and many other organisms. Their experimental procedures will provide the first understanding of how full-length PAM binds to biologically relevant neuropeptide precursors. In addition, their work will provide new insights into the interaction of PAM and the molecular precursor for atrial natriuretic peptide (pro-ANP), setting the scene for discovering why PAM is present in tissues that do not make neuropeptides. The project will provide training for a number of graduate, undergraduate, and high school students in the use of a broad array of modern biochemical methods. The results of these studies will form the basis for a series of interactive, in-person talks at the Museum of Science and Industry (MOSI), located close to the USF campus, that will be tailored for students in grades 3-5, 6-8, or 9-12, and members of the general public. These talks will aim to not only improve scientific literacy in Florida but also inspire young people to pursue scientific research in neuroscience and medicine. This research project will provide a detailed molecular understanding of how the full-length, bifunctional form of PAM is able to recognize and modify a wide range of neuropeptide precursors as substrates. They will also provide the first information about how the dynamical properties of PAM impact both catalytic mechanism and communication between the two active sites, which are located in different domains of the enzyme. In addition, we will explore the structural basis of how bifunctional PAM binds to pro-ANP, a peptide that is not a substrate for the enzyme. Our studies will yield the first molecular understanding of the PAM/pro-ANP complex, test current hypotheses about the importance of this complex for cells in tissues that do not make or secrete α-amidated peptides. These project objectives will be accomplished using a multidisciplinary approach that combines biochemical assays, peptide synthesis, hydrogen/deuterium exchange (HDX) measurements, computational modeling, and cryogenic electron microscopy (cryo-EM). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.