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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., (© 2019 Federation of European Biochemical Societies.)

Peptides and proteins possess an inherent tendency to self-assemble, prompting the formation of amyloid aggregates from their soluble and functional states. Amyloids are linked to many devastating diseases, but self-assembling proteins can also represent formidable tools to produce new and sustainable biomaterials for biomedical and biotechnological applications. The mechanism of fibrillar aggregation, which influences the morphology and the properties of the protein aggregates, depend on factors such as pH, ionic strength, temperature, agitation, and protein concentration. We have here used intensive mechanical agitation, with or without beads, to prompt the aggregation of the single-chain derivative of the plant protein monellin, named MNEI, which is a well characterized sweet protein. Transmission electron microscopy confirmed the formation of fibrils several micrometers long, morphologically different from the previously characterized fibers of MNEI. Changes in the protein secondary structures during the aggregation process were monitored by Fourier transform infrared spectroscopy, which detected differences in the conformation of the final aggregates obtained under mechanical agitation. Moreover, soluble oligomers could be detected in the early phases of aggregation by polyacrylamide gel-electrophoresis. These findings emphasize the existence of multiple pathways of fibrillar aggregation for MNEI, which could be exploited for the design of innovative protein-based biomaterials., (Copyright © 2020. Published by Elsevier B.V.)

Background: MNEI and its variant Y65R-MNEI are sweet proteins with potential applications as sweeteners in food industry. Also, they are often used as model systems for folding and aggregation studies., Methods: X-ray crystallography was used to structurally characterize Y65R-MNEI at five different pHs, while circular dichroism and fluorescence spectroscopy were used to study their thermal and chemical stability. ThT assay and AFM were used for studying the kinetics of aggregation and morphology of the aggregates., Results: Crystal structures of Y65R-MNEI revealed the existence of a dimer in the asymmetric unit, which, depending on the pH, assumes either an open or a closed conformation. The pH dramatically affects kinetics of formation and morphology of the aggregates: both MNEI and Y65R-MNEI form fibrils at acidic pH while amorphous aggregates are observed at neutral pH., Conclusions: The mutation Y65R induces structural modifications at the C-terminal region of the protein, which account for the decreased stability of the mutant when compared to MNEI. Furthermore, the pH-dependent conformation of the Y65R-MNEI dimer may explain the different type of aggregates formed as a function of pH., General Significance: The investigation of the structural bases of aggregation gets us closer to the possibility of controlling such process, either by tuning the physicochemical environmental parameters or by site directed mutagenesis. This knowledge is helpful to expand the range of stability of proteins with potential industrial applications, such as MNEI and its mutant Y65R-MNEI, which should ideally preserve their structure and soluble state through a wide array of conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sweet proteins are a class of proteins with the ability to elicit a sweet sensation in humans upon interaction with sweet taste receptor T1R2/T1R3. Single-chain Monellin, MNEI, is among the sweetest proteins known and it could replace sugar in many food and beverage recipes. Nonetheless, its use is limited by low stability and high aggregation propensity at neutral pH. To solve this inconvenience, we designed a new construct of MNEI, dubbed Mut9, which led to gains in both sweetness and stability. Mut9 showed an extraordinary stability in acidic and neutral environments, where we observed a melting temperature over 20 °C higher than that of MNEI. In addition, Mut9 resulted twice as sweet than MNEI. Both proteins were extensively characterized by biophysical and sensory analyses. Notably, Mut9 preserved its structure and function even after 10 min boiling, with the greatest differences being observed at pH 6.8, where it remained folded and sweet, whereas MNEI lost its structure and function. Finally, we performed a 6-month shelf-life assessment, and the data confirmed the greater stability of the new construct in a wide range of conditions. These data prove that Mut9 has an even greater potential for food and beverage applications than MNEI. [ABSTRACT FROM AUTHOR]