Prion diseases are infectious and fatal neurodegenerative disorders with no available treatment. Zoonotic prion transmission is of concern, as the bovine spongiform encephalopathy (BSE) epidemic has led to nearly 200 cases of prion infection in humans. New prion diseases have recently emerged in food animals and wild deer with unknown potential for spread to humans. The molecular mechanisms that underlie prion aggregation, transmission between species, and the prion strains are still poorly understood. Our long term goal is to identify the key residues of the prion protein that govern species barriers and strain conformation. The 2-a2 loop of the prion protein (amino acids 165-175) is a site of exceptionally high sequence variability. Microcrystallography has shown that adjacent 2-a2 loops can align as -sheets with side chains that intermesh in a dry interface as part of the amyloid core. We have shown that two amino acid substitutions in the 2-a2 loop of the mouse prion protein lead to de novo prion disease in transgenic mice. In preliminary studies, these substitutions markedly change the interspecies transmission barriers in mice. Here we propose that the 2-a2 loop also plays an important role for prion transmission to humans and cattle. In this proposal, we will evaluate the impact of the 2-a2 loop residues on prion aggregation, species barriers, and strains. In Aim 1, we will determine the role of critical residues for prion aggregation and conformational conversion in vitro and in vivo. In Aim 2, we will assess the importance of the 2-a2 loop region for prion infection of humans and cattle using mouse models that differ only at the loop. Results from these studies will contribute to our understanding of the fundamental mechanisms of prion protein aggregation, aid in our risk assessment of prion transmission to humans, as well as advance the rational design of therapeutics to block prion aggregation.