Résumé : Cystic fibrosis (CF) is the most common lethal genetic disease in Western countries.It is due to mutations in the gene coding for the cystic fibrosis transmembraneregulator (CFTR), a chloride channel required for proper fluid balance in many tissues(lungs, digestive organs, etc.). In over 70% of the CF alleles, the disease-causingmutation is the deletion of the phenylalanine 508 (F508del), located in the nucleotidebinding domain 1 (NBD1). In our recent published study, a novel conformationof NBD1 (called the β-SS conformation) was captured using a conformation-specificnanobody. This β-SS conformation and the canonical conformation of NBD1 (theconformation previously described in the literature) interchange following an equilibriumtightly correlated to stability and function (destabilising conditions suchas F508del mutation disfavour the canonical conformation). This study indicatedthat the transition between the conformations is an Achille’s heel in the structurallandscape of the protein and enables protein unfolding by the prevalent pathologicalmutation F508del.The present research focused on investigating if this correlation between plasticityand pathology is unique for F508del or also observed for other disease-causingmutations of NBD1 as this observation could lead to the development of therapiescommon to several untreated NBD1 mutations. To measure if rare mutants of NBD1shifted the equilibrium towards the β-SS states, we followed the conformationallandscape of NBD1 by single molecule fluorescence (smFRET), specifically thetransitions between the β-SS and the canonical conformation. In addition, we usedThermal Shift Assay (TSA) to test the thermostability of those mutants with differentconditions known to affect the equilibrium and stability of F508del such as ATP,mutations, or a promising stabilising nanobody. Finally, we crystallised some mutantsto look for any structural changes.We show that all mutations tested decreased the canonical population in FRETmeasurements. TSA analysis showed that all the mutants are thermally destabilisedand can be stabilised with the same conditions as for F508del. Using a series ofRIdel variants we showed by X-ray crystallography that the mutations do not preventfolding of the domain. Therefore, we conclude that, mutations studied in NBD1,enable the premature unfolding of the protein not by disrupting structural elementsbut rather by perturbing structural transitions between the canonical and the β-SSconformation. This work provides a new framework to design conformation-basedtherapeutics for several mutations lacking any treatment so far.