Abstract Neurofibromin is the 320-kilodalton protein product of the NF1 gene that is mutated in the Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to abrogation of the GTPase-activating activity of the protein, leading to aberrant signaling through the RAS-MAPK pathway. Very little is known about the function of the majority of the NF1 protein—to date, only the GAP domain and a region containing a Sec-PH motif have detailed structural information. To better understand the role of this large protein, we have carried out a series of biochemical and biophysical studies that clearly show that full-length NF1 protein exists primarily as a dimer. Data from in vivo experiments confirmed that this dimerization also occurs in cells, and presumably has biologic implications. A model was generated from negative-stain EM data that suggests that NF1 monomers form both intermolecular contacts to form dimers and intramolecular contacts that stabilize the individual monomers. Deletion analysis permitted us to map these two interacting domains to different parts of the NF1 protein, and crosslinking studies coupled with mass spectrometry helped to identify regions of possible interaction. In addition, introduction of point mutations observed in patients with NF diseases caused a dramatic decrease in the stability of proteins both in vitro and in vivo, potentially arguing for a rationale for loss of NF1 activity by these single mutations. This abstract is also being presented as Poster A03. Citation Format: Mukul Sherekar, Sae-Won Han, Simon Messing, Matthew Drew, Xiaoying Ye, William Gillette, Puneet Juneja, Hugh O’Neill, Christopher Stanley, Arvind Ramanathan, Natalia de Val, Claire Lorenzo, Frank McCormick, Dominic Esposito. Biochemical and structural analysis of the neurofibromin (NF1) protein and a potential role for protein destabilization in Rasopathy diseases [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR08.