Douglas V. Laurents, Diletta Ami, Masoud Delfi, Federica Donnarumma, Delia Picone, Alessandro Emendato, Serena Leone, Roberta Spadaccini, Antonino Natalello, Donnarumma, F, Leone, S, Delfi, M, Emendato, A, Ami, D, Laurents, D, Natalello, A, Spadaccini, R, Picone, D, Ministerio de Economía y Competitividad (España), European Commission, Università degli Studi di Milano-Bicocca, Consejo Superior de Investigaciones Científicas (España), Ministero dell'Istruzione, dell'Università e della Ricerca, Donnarumma, F., Leone, S., Delfi, M., Emendato, A., Ami, D., Laurents, D. V., Natalello, A., Spadaccini, R., and Picone, D.
15 pags., 11 figs., Protein self-assembly is a ubiquitous phenomenon, traditionally studied for its links to amyloid pathologies, which has also gained attention as its physiological roles and possible biotechnological applications emerged over time. It is also known that varying the conditions to which proteins are exposed can lead to aggregate polymorphism. To understand the factors that trigger aggregation and/or direct it toward specific outcomes, we performed a multifaceted structural characterization of the thermally induced self-assembly process of MNEI, a model protein able to form amyloid aggregates under nondenaturing conditions. MNEI is also known for its extreme sweetness which, combined with a considerable thermal stability, makes the protein a promising alternative sweetener. Fourier-transformed infrared spectroscopy and electron microscopy data showed that the presence of NaCl accelerates the kinetics of fibrillar aggregation, while disfavoring the population of off-pathway states that are instead detected by native gel electrophoresis at low ionic strength. NMR studies revealed how NaCl modulates the self-assembling mechanism of MNEI, switching the process from soluble oligomeric forms to fibrils. Comparative analysis demonstrated that the presence of NaCl induces local differences in the protein dynamics and surface accessibility, without altering the native fold. We identified the regions most affected by the presence of NaCl, which control the aggregation process, and represent hot spots on the protein surface for the rational design of new mutants with controlled aggregation propensity., This work was partially supported by Fondazione conil SUD (grant 2011-PDR-19) and SAF2016-76678-C2-2-R from the Spanish Ministry of Economy and Competitivity. We acknowledge the Horizon 2020 Pro-gramme (grant agreement No. 653706, iNEXT) and University of Milano-Bicocca (grant ‘Fondo di Ateneoper la Ricerca 2017-ATE-0339’). Some NMR experiments were performed in the ‘Manuel Rico’ NMR lab-oratory (LMR) of the Spanish National ResearchCouncil (CSIC), a node of the Spanish Large-ScaleNational Facility (ICTS R-LRB). FD was the recipientof a fellowship from Regione Campania (POR Campania FSE 2014–2020). MD was supported by PON CCI2014IT16M2OP005 from the Italian Ministry ofUniversity and Research