The dentin-enamel junction (DEJ) in teeth provide a strong and tough interface that can withstand a long working life with little to no remodeling. However, the molecular composition and structure that leads to these remarkable mechanical properties are still poorly understood. Specifically, how proteins from the enamel matrix interact with dentin collagen fibrils to form a continuously mineralized tissue, needs to be addressed. The goal of this project is to advance our understanding of the nanoscale origin of the strength and toughness of the DEJ. We propose to define the role of collagen and amelogenin, the major proteins from dentin and enamel respectively, on the strength of DEJ through two specific aims. In the first aim we will test that amelogenin is not required for the extension of mineral crystals from dentin’s collagen, while preventing the cleavage of the full-length protein alters the structure of the DEJ. In the second aim we will demonstrate that mutations in dentin collagen can affect the formation and mechanical properties of the DEJ. Our first hypothesis is supported by the fact that the DEJ of amelogenin knock-out mice appears intact while mouse models lacking matrix metalloproteinase-20 (MMP-20), the main enzyme responsible for the cleavage of enamel proteins during enamel secretion, have a compromised DEJ and enamel delaminates from the dentin. In our second aim, we will explore how mutation of type I collagen in mouse models of dentinogenesis imperfecta alter the DEJ. We will test these hypotheses by using high-resolution electron microscopy and immunohistochemistry techniques to characterize in situ the morphology of DEJ as well as the organic matrix composition in wild type and mutant mice. Similarly, we will explore the organic matrix nanostructure in relation to mineral organization and crystallinity. We will also assess the impact of collagen mutation on its affinity to enamel proteins in vitro. Finally, we will quantify the impact of mutations on the strength and toughness of the DEJ using innovative atomic force microscopy characterization. Taken together, the results of this project will significantly improve our understanding of the DEJ and on the mechanisms leading to its incredible mechanical behavior.