The isolated GTPase-activating-protein-related domain of neurofibromin-1 has a low conformational stability in solution Neira, José Luis Vega, Sonia Martínez Rodríguez, Sergio Velázquez Campoy, Adrián Circular Dichroism Conformational stability Differential scanning calorimetry This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN, BFU2016-78232-P to AVC]; Andalusian Regional Government [“Endocrinology and Metabolism Group” CTS202 to SMR]; Diputacion ´ General de Aragon ´ [“Protein targets and Bioactive Compounds group” E45-20R to AVC]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Neurofibromin-1 (NF1) is a large, multidomain tumour suppressor encoded by the NF1 gene. The gene is mutated in neurofibromatosis type I, a disease characterized by malignant tumours of the nervous system and benign neurofibromas. The best-known activity of NF1 is the down-regulation of the mitogen-activated protein kinase pathway via its three-hundred-residue-long GTPase-activating protein (GAP) domain (the so-called GAP-related domain (NF1-GRD)). The NF1-GRD stimulates Ras GTPase activity in turning off signalling. Despite this activity, NF1-GRD has been demonstrated to bind to other different proteins, such as SPRED1 or MC1R. We have embarked on the biophysical and conformational characterization of NF1-GRD in solution by using several spectroscopic (namely fluorescence and circular dichroism (CD)) and biophysical techniques (namely size exclusion chromatography (SEC) and differential scanning calorimetry (DSC)). This biophysical characterization is crucial in deciphering NF1-GRD interactome and in finding biochemical features, modulating possible protein interactions. The native-like structure of NF1-GRD (as monitored by intrinsic fluorescence and far-UV CD) was strongly pH-dependent showing a pH-titration causing a substantial increase in its helicity. NF1-GRD had a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by intrinsic and ANS fluorescence, and CD. Chemical denaturations showed that NF1-GRD unfolded through an intermediate which has a substantial amount of solvent-exposed hydrophobic patches. 2025-12-16T09:57:05Z 2025-12-16T09:57:05Z 2021 journal article Neira, J. L., Vega, S., Martínez-Rodríguez, S., & Velázquez-Campoy, A. (2021). The isolated GTPase-activating-protein-related domain of neurofibromin-1 has a low conformational stability in solution. Archives of biochemistry and biophysics, 700, 108767. https://doi.org/10.1016/j.abb.2021.108767 0003-9861 https://hdl.handle.net/10481/108846 10.1016/j.abb.2021.108767 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional Elsevier