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Göran Hellekant - One of the best experts on this subject based on the ideXlab platform.
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Monkey Electrophysiological and Human Psychophysical Responses to Mutants of the Sweet Protein Brazzein: Delineating Brazzein Sweetness
2015Co-Authors: Zheyuan Jin, Fariba M. Assadi-porter, John L. Markley, Göran HellekantAbstract:Responses to brazzein, 25 brazzein mutants and two forms of monellin were studied in two types of experiments: electrophysiological recordings from chorda tympani S fibers of the rhesus monkey, Macaca mulatta, and psychophysical experiments. We found that different mutations at position 29 (changing Asp29 to Ala, Lys or Asn) made the molecule significantly Sweeter than brazzein, while mutations at positions 30 or 33 (Lys30Asp or Arg33Ala) removed all Sweetness. The same pattern occurred again at the β-turn region, where Glu41Lys gave the highest Sweetness score among the mutants tested, whereas a mutation two residues distant (Arg43Ala) abolished the Sweetness. The effects of charge and side chain size were examined at two locations, namely positions 29 and 36. The findings indicate that charge is important for eliciting Sweetness, whereas the length of the side-chain plays a lesser role. We also found that the N- and C-termini are important for the Sweetness of brazzein. The close correlation (r = 0.78) between the results of the above two methods corroborates our hypothesis that S fibers convey Sweet taste in primates. Key words: low-caloric natural Sweeteners, Magnitude Labeled Scale, rhesus monkey, Sweet taste recepto
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Design and Evaluation of New Analogs of the Sweet Protein Brazzein
Chemical senses, 2009Co-Authors: D. Eric Walters, Zheyuan Jin, Tiffany Cragin, Jon N. Rumbley, Göran HellekantAbstract:We have previously modeled the interaction of the Sweet Protein brazzein with the extracellular domains of the Sweet taste receptor. Here, we describe the application of that model to the design of 12 new highly potent analogs of brazzein. Eight of the 12 analogs have higher Sweetness potency than wild-type brazzein. Results are consistent with our brazzein–receptor interaction model. The model predicts binding of brazzein to the open form of T1R2 in the T1R2–T1R3 heterodimer.
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Interactions of the Sweet Protein Brazzein with the Sweet Taste Receptor
Journal of agricultural and food chemistry, 2006Co-Authors: D. Eric Walters, Göran HellekantAbstract:Brazzein is a small, potently Sweet Protein. Homology modeling has been used to construct a model of the ligand-binding domain of the Sweet taste receptor, and low-resolution docking has been used to identify potential modes of brazzein−receptor binding. Published brazzein mutation−taste data were then used to select one of these as the most likely brazzein−receptor binding orientation. This orientation places brazzein in contact primarily with the T1R2 subunit of the receptor, and it accounts for 21 of the 23 mutation results examined. Keywords: Brazzein; Sweetener; taste receptor; homology model; docking; Pentadiplandra brazzeana
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probing the Sweet determinants of brazzein wild type brazzein and a tasteless variant brazzein ins r18a i18b exhibit different ph dependent nmr chemical shifts
Biochemical and Biophysical Research Communications, 2005Co-Authors: Qin Zhao, Vicktoria Danilova, Göran Hellekant, Zheyuan Jin, Jikui Song, John L. MarkleyAbstract:Brazzein is a small, intensely Sweet Protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg-Ile) were inserted between Leu18 and Ala19 of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single alpha-helix, and around the Cys16-Cys37 disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the alpha-helix and the second beta-strand. In particular, several residues of brazzein previously shown to be essential for its Sweetness (His31, Arg33, Glu41, Arg43, Asp50, and Tyr54) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His31, Glu41, Asp50, and Tyr54 were unaltered by the insertion. The insertion led to large chemical shift and pKa perturbation of Glu36, a residue shown previously to be important for brazzein's Sweetness. These results serve to refine the known Sweetness determinants of brazzein and lend further support to the idea that the Protein interacts with a Sweet-taste receptor through a multi-site interaction mechanism, as has been postulated for brazzein and other Sweet Proteins (monellin and thaumatin).
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Brazzein a Small, Sweet Protein: Discovery and Physiological Overview
Chemical Senses, 2005Co-Authors: Göran Hellekant, Vicktoria DanilovaAbstract:, 1968; Kurihara and Beidler, 1968) amol. wt of 24 600.The search for Sweeteners was no longer limited to small moleculesand resulted within a few years in the discovery of monellin andthaumatin (Morris and Cagan, 1972; van der Wel and Loeve, 1972).Later, mabinlin and curculin were discovered (Hu and He, 1983;Yamashita
Kwang-hoon Kong - One of the best experts on this subject based on the ideXlab platform.
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3M-Brazzein as a Natural Sugar Substitute Attenuates Obesity, Metabolic Disorder, and Inflammation
Journal of agricultural and food chemistry, 2020Co-Authors: Hansaem Kim, Kwang-hoon Kong, Byung-ha Kang, Jaeyong Kang, Seungwoo Hong, Hyangsoon Noh, Suhyun Park, Young-jin Seo, Sungguan HongAbstract:Obesity is a global chronic disease linked to various diseases. Increased consumption of added sugars, especially in beverages, is a key contributor to the obesity epidemic. It is essential to reduce or replace sugar intake with low-calorie Sweeteners. Here, a natural Sweet Protein, 3M-brazzein, was investigated as a possible sugar substitute. Mice were exposed to 3M-brazzein or 10% sucrose of equivalent Sweetness, in drinking water to mimic human obesity development over 15 weeks. Consumption of 3M-brazzein in liquid form did not cause adiposity hypertrophy, resulting in 33.1 ± 0.4 g body weight and 0.90 ± 0.2 mm fat accumulation, which were 35.9 ± 0.7 g (p = 0.0094) and 1.53 ± 0.067 mm (p = 0.0031), respectively, for sucrose supplement. Additionally, 3M-brazzein did not disrupt glucose homeostasis or affect insulin resistance and inflammation. Due to its naturally low-calorie content, 3M-brazzein could also be a potential sugar substitute that reduces adiposity.
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improved secretory production of the Sweet tasting Protein brazzein in kluyveromyces lactis
Journal of Agricultural and Food Chemistry, 2016Co-Authors: Cho-rong Yun, Ji-na Kong, Ju-hee Chung, Myung-chul Kim, Kwang-hoon KongAbstract:Brazzein is an intensely Sweet Protein with high stability over a wide range of pH values and temperatures, due to its four disulfide bridges. Recombinant brazzein production through secretory expression in Kluyveromyces lactis is reported, but is inefficient due to incorrect disulfide formation, which is crucial for achieving the final Protein structure and stability. Protein disulfide bond formation requires Protein disulfide isomerase (PDI) and Ero1p. Here, we overexpressed KlPDI in K. lactis or treated the cells with dithiothreitol to overexpress KlERO1 and improve brazzein secretion. KlPDI and KlERO1 overexpression independently increased brazzein secretion in K. lactis by 1.7-2.2- and 1.3-1.6-fold, respectively. Simultaneous overexpression of KlPDI and KlERO1 accelerated des-pE1M-brazzein secretion by approximately 2.6-fold compared to the previous system. Moreover, intracellular misfolded/unfolded recombinant des-pE1M-brazzein was significantly decreased. In conclusion, increased KlPDI and KlERO1 expression favors brazzein secretion, suggesting that correct Protein folding may be crucial to brazzein secretion in K. lactis.
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Efficient secretory expression of the Sweet-tasting Protein brazzein in the yeast Kluyveromyces lactis
Protein expression and purification, 2013Co-Authors: Jin-seok Noh, Kwang-hoon KongAbstract:Abstract Brazzein is an intensely Sweet-tasting Protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate Sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a Sweet Protein in the yeast Kluyveromyces lactis . The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomyces cerevisiae α-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96 h at 30 °C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104 mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS–PAGE, HPLC, and circular dichroism. The identity of the recombinant Protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic Sweetness in its minor form, approximately 2130 times Sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry.
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Multiple mutations of the critical amino acid residues for the Sweetness of the Sweet-tasting Protein, brazzein.
Food chemistry, 2012Co-Authors: Joo-won Lee, Ji-eun Cha, Kwang-hoon KongAbstract:We have previously identified critical residues important for Sweetness of the Sweet Protein brazzein by site-directed mutagenesis (Yoon, Kong, Jo, & Kong, 2011). In order to elucidate the interaction mechanisms of brazzein with the Sweet taste receptor, we made multiple mutations of three residues (His31 in loop 30-33, Glu36 in β-strand III, and Glu41 in loop 40-43). We found that all double mutations (H31R/E36D, H31R/E41A and E36D/E41A) made the molecules Sweeter than des-pE1M-brazzein and three single mutants. Moreover, the triple mutation (H31R/E36D/E41A) made the molecule significantly Sweeter than three double mutants. These results strongly support the hypothesis that brazzein binds to the multisite surface of the Sweet taste receptor. Our findings also suggest that mutations reducing the overall negative charge and/or increasing the positive charge favour Sweet-tasting Protein potency.
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Residue mutations in the Sweetness loops for the Sweet-tasting Protein brazzein
Food Chemistry, 2011Co-Authors: Sug-young Yoon, Ji-na Kong, Kwang-hoon KongAbstract:Abstract To identify critical residues, important for Sweetness, of the Sweet Protein brazzein, 11 mutants of the residues in three loops of brazzein were constructed by site-directed mutagenesis. We found that mutations of Glu41 to Ala, Lys, or Arg at position 41 in loop 40–43 made the molecules significantly Sweeter than brazzein, while mutations at two distant residues (changing Arg43 to Lys or Glu) decreased Sweetness. A similar pattern occurred at loop 30–33, where mutation of the His31 to Arg significantly increased Sweetness, while mutations at positions 30 or 33 in the immediate vicinity of this region significantly decreased Sweetness. In addition, a Gln17 residue in the loop 9–19 was necessary for structural integrity. From these results, we suggest that the loops containing His31 and Glu41 are critical regions of the molecule for eliciting Sweetness, and the charge and/or structure of the side chain of these residues play an important role in the multi-point interactions between brazzein and the Sweet-taste receptor.
John L. Markley - One of the best experts on this subject based on the ideXlab platform.
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Monkey Electrophysiological and Human Psychophysical Responses to Mutants of the Sweet Protein Brazzein: Delineating Brazzein Sweetness
2015Co-Authors: Zheyuan Jin, Fariba M. Assadi-porter, John L. Markley, Göran HellekantAbstract:Responses to brazzein, 25 brazzein mutants and two forms of monellin were studied in two types of experiments: electrophysiological recordings from chorda tympani S fibers of the rhesus monkey, Macaca mulatta, and psychophysical experiments. We found that different mutations at position 29 (changing Asp29 to Ala, Lys or Asn) made the molecule significantly Sweeter than brazzein, while mutations at positions 30 or 33 (Lys30Asp or Arg33Ala) removed all Sweetness. The same pattern occurred again at the β-turn region, where Glu41Lys gave the highest Sweetness score among the mutants tested, whereas a mutation two residues distant (Arg43Ala) abolished the Sweetness. The effects of charge and side chain size were examined at two locations, namely positions 29 and 36. The findings indicate that charge is important for eliciting Sweetness, whereas the length of the side-chain plays a lesser role. We also found that the N- and C-termini are important for the Sweetness of brazzein. The close correlation (r = 0.78) between the results of the above two methods corroborates our hypothesis that S fibers convey Sweet taste in primates. Key words: low-caloric natural Sweeteners, Magnitude Labeled Scale, rhesus monkey, Sweet taste recepto
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Temperature-dependent conformational change affecting Tyr11 and Sweetness loops of brazzein.
Proteins, 2013Co-Authors: Claudia C. Cornilescu, John L. Markley, Marco Tonelli, Hongyu Rao, Gabriel Cornilescu, Sarah F. Porter, Michele L. Derider, Fariba M. Assadi-porterAbstract:The Sweet Protein brazzein, a member of the Csβα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the Protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side-chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the Sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the Sweetness of brazzein, may represent the bound state of brazzein in the complex with the human Sweet receptor.
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Key amino acid residues involved in multi-point binding interactions between brazzein, a Sweet Protein, and the T1R2-T1R3 human Sweet receptor.
Journal of molecular biology, 2010Co-Authors: Fariba M. Assadi-porter, John L. Markley, Emeline Maillet, Jeniffer Quijada, James T. Radek, Marianna MaxAbstract:The Sweet Protein brazzein [recombinant Protein with sequence identical with the native Protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human Sweet receptor, a heterodimeric G-Protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the Sweet receptor):R217A/hT1R3 (human T1R3 subunit of the Sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the Sweet receptor by some mechanism other than the proposed wedge models.
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How Sweet It Is : Detailed Molecular and Functional Studies of Brazzein, a Sweet Protein and Its Analogs
2008Co-Authors: Fariba M. Assadi-porter, Marco Tonelli, James T. Radek, Claudia C. Cornilescu, John L. MarkleyAbstract:Brazzein is a small, low-calorie, Sweet Protein with high stability over wide temperature and pH ranges. Brazzein has desirable taste characteristics that resemble those of carbohydrate Sweeteners. Brazzein folds in a β-α-β 2 topology in which the α-helix packs against the three-stranded antiparallel β-sheet. This structure is held together by four disulfide bridges. We developed an efficient bacterial production system for brazzein that allows us to express wild-type and mutant Proteins. We have designed a large number of brazzein variants for taste tests. These include mutations that affect surface charges, disulfide bridges, loops, and flexible regions. We have subjected a subset of these variants to detailed analysis by NMR spectroscopy to identify patterns of hydrogen bonds and internal mobility. The results show a correlation between these physical properties and the Sweetness of the Protein. These results led us to propose a multi-site binding model for the interaction between brazzein and the heterodimeric human Sweet receptor, which we are continuing to test with the goal of designing more potent brazzein analogs as potential future Sweeteners.
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Efficient and rapid Protein expression and purification of small high disulfide containing Sweet Protein brazzein in E. coli
Protein expression and purification, 2007Co-Authors: Fariba M. Assadi-porter, Sammy Patry, John L. MarkleyAbstract:Brazzein Protein comes from an edible fruit, which has a long history of being a staple in the local human diet in Africa. The attractive features of brazzein as a potential commercial Sweetener include its small size (53 amino acid residues), its stability over wide ranges of temperature and pH, and the similarity of its Sweetness to sucrose. Heterologous production of brazzein is complicated by the fact that the Protein contains four disulfide bridges and requires a specific N-terminal sequence. Our previous protocol for producing the Protein from Escherichia coli involved several steps with low overall yield: expression as a fusion Protein, denaturation and renaturation, oxidation of the cysteines, and cleavage by cyanogen bromide at an engineered methionine adjacent to the desired N-terminus. The new protocol described here, which is much faster and leads to a higher yield of native Protein, involves the production of brazzein in E. coli as a fusion with SUMO. The isolated Protein product contains the brazzein domain folded with correct disulfide bonds formed and is then cleaved with a specific SUMO protease to liberate native brazzein. This protocol represents an important advancement that will enable more efficient research into the interaction between brazzein and the receptor as well as investigations to test the potential of brazzein as a commercially viable natural low calorie Sweetener.
Delia Picone - One of the best experts on this subject based on the ideXlab platform.
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Getting value from the waste: recombinant production of a Sweet Protein by Lactococcus lactis grown on cheese whey
BMC, 2018Co-Authors: Mohamed Boumaiza, Serena Leone, Delia Picone, Andrea Colarusso, Ermenegilda Parrilli, Elena Garcia-fruitós, Angela Casillo, Anna Arís, Maria Michela Corsaro, Maria Luisa TutinoAbstract:Abstract Background Recent biotechnological advancements have allowed for the adoption of Lactococcus lactis, a typical component of starter cultures used in food industry, as the host for the production of food-grade recombinant targets. Among several advantages, L. lactis has the important feature of growing on lactose, the main carbohydrate in milk and a majoritarian component of dairy wastes, such as cheese whey. Results We have used recombinant L. lactis NZ9000 carrying the nisin inducible pNZ8148 vector to produce MNEI, a small Sweet Protein derived from monellin, with potential for food industry applications as a high intensity Sweetener. We have been able to sustain this production using a medium based on the cheese whey from the production of ricotta cheese, with minimal pre-treatment of the waste. As a proof of concept, we have also tested these conditions for the production of MMP-9, a Protein that had been previously successfully obtained from L. lactis cultures in standard growth conditions. Conclusions Other than presenting a new system for the recombinant production of MNEI, more compliant with its potential applications in food industry, our results introduce a strategy to valorize dairy effluents through the synthesis of high added value recombinant Proteins. Interestingly, the possibility of using this whey-derived medium relied greatly on the choice of the appropriate codon usage for the target gene. In fact, when a gene optimized for L. lactis was used, the production of MNEI proceeded with good yields. On the other hand, when an E. coli optimized gene was employed, Protein synthesis was greatly reduced, to the point of being completely abated in the cheese whey-based medium. The production of MMP-9 was comparable to what observed in the reference conditions
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ph driven fibrillar aggregation of the super Sweet Protein y65r mnei a step by step structural analysis
Biochimica et Biophysica Acta, 2017Co-Authors: Andrea Pica, Serena Leone, Michele F Rega, Federica Donnarumma, Rocco Di Girolamo, Alessandro Emendato, Antonello Merlino, Delia PiconeAbstract:Abstract 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.
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molecular dynamics driven design of ph stabilized mutants of mnei a Sweet Protein
PLOS ONE, 2016Co-Authors: Serena Leone, Delia PiconeAbstract:MNEI is a single chain derivative of monellin, a plant Protein that can interact with the human Sweet taste receptor, being therefore perceived as Sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible Protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the Protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.
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acetate friend or foe efficient production of a Sweet Protein in escherichia coli bl21 using acetate as a carbon source
Microbial Cell Factories, 2015Co-Authors: Serena Leone, Ermenegilda Parrilli, Maria Luisa Tutino, Filomena Sannino, Delia PiconeAbstract:Escherichia coli is, to date, the most used microorganism for the production of recombinant Proteins and biotechnologically relevant metabolites. High density cell cultures allow efficient biomass and Protein yields. However, their main limitation is the accumulation of acetate as a by-product of unbalanced carbon metabolism. Increased concentrations of acetate can inhibit cellular growth and recombinant Protein production, and many efforts have been made to overcome this problem. On the other hand, it is known that E. coli is able to grow on acetate as the sole carbon source, although this mechanism has never been employed for the production of recombinant Proteins. By optimization of the fermentation parameters, we have been able to develop a new acetate containing medium for the production of a recombinant Protein in E. coli BL21(DE3). The medium is based on a buffering phosphate system supplemented with 0.5% yeast extract for essential nutrients and sodium acetate as additional carbon source, and it is compatible with lactose induction. We tested these culture conditions for the production of MNEI, a single chain derivative of the Sweet plant Protein monellin, with potential for food and beverage industries. We noticed that careful oxygenation and pH control were needed for efficient Protein production. The expression method was also coupled to a faster and more efficient purification technique, which allowed us to obtain MNEI with a purity higher than 99%. The method introduced represents a new strategy for the production of MNEI in E. coli BL21(DE3) with a simple and convenient process, and offers a new perspective on the capabilities of this microorganism as a biotechnological tool. The conditions employed are potentially scalable to industrial processes and require only low-priced reagents, thus dramatically lowering production costs on both laboratory and industrial scale. The yield of recombinant MNEI in these conditions was the highest to date from E. coli cultures, reaching on average ~180 mg/L of culture, versus typical LB/IPTG yields of about 30 mg/L.
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design of Sweet Protein based Sweeteners hints from structure function relationships
Food Chemistry, 2015Co-Authors: Michele F Rega, Serena Leone, Rossella Di Monaco, Silvana Cavella, Federica Donnarumma, Roberta Spadaccini, Delia PiconeAbstract:Sweet Proteins represent a class of natural molecules, which are extremely interesting regarding their potential use as safe low-calories Sweeteners for individuals who need to control sugar intake, such as obese or diabetic subjects. Punctual mutations of amino acid residues of MNEI, a single chain derivative of the natural Sweet Protein monellin, allow the modulation of its taste. In this study we present a structural and functional comparison between MNEI and a Sweeter mutant Y65R, containing an extra positive charge on the Protein surface, in conditions mimicking those of typical beverages. Y65R exhibits superior Sweetness in all the experimental conditions tested, has a better solubility at mild acidic pH and preserves a significant thermal stability in a wide range of pH conditions, although slightly lower than MNEI. Our findings confirm the advantages of structure-guided Protein engineering to design improved low-calorie Sweeteners and excipients for food and pharmaceutical preparations.
Fariba M. Assadi-porter - One of the best experts on this subject based on the ideXlab platform.
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Monkey Electrophysiological and Human Psychophysical Responses to Mutants of the Sweet Protein Brazzein: Delineating Brazzein Sweetness
2015Co-Authors: Zheyuan Jin, Fariba M. Assadi-porter, John L. Markley, Göran HellekantAbstract:Responses to brazzein, 25 brazzein mutants and two forms of monellin were studied in two types of experiments: electrophysiological recordings from chorda tympani S fibers of the rhesus monkey, Macaca mulatta, and psychophysical experiments. We found that different mutations at position 29 (changing Asp29 to Ala, Lys or Asn) made the molecule significantly Sweeter than brazzein, while mutations at positions 30 or 33 (Lys30Asp or Arg33Ala) removed all Sweetness. The same pattern occurred again at the β-turn region, where Glu41Lys gave the highest Sweetness score among the mutants tested, whereas a mutation two residues distant (Arg43Ala) abolished the Sweetness. The effects of charge and side chain size were examined at two locations, namely positions 29 and 36. The findings indicate that charge is important for eliciting Sweetness, whereas the length of the side-chain plays a lesser role. We also found that the N- and C-termini are important for the Sweetness of brazzein. The close correlation (r = 0.78) between the results of the above two methods corroborates our hypothesis that S fibers convey Sweet taste in primates. Key words: low-caloric natural Sweeteners, Magnitude Labeled Scale, rhesus monkey, Sweet taste recepto
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Temperature-dependent conformational change affecting Tyr11 and Sweetness loops of brazzein.
Proteins, 2013Co-Authors: Claudia C. Cornilescu, John L. Markley, Marco Tonelli, Hongyu Rao, Gabriel Cornilescu, Sarah F. Porter, Michele L. Derider, Fariba M. Assadi-porterAbstract:The Sweet Protein brazzein, a member of the Csβα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the Protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side-chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the Sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the Sweetness of brazzein, may represent the bound state of brazzein in the complex with the human Sweet receptor.
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Key amino acid residues involved in multi-point binding interactions between brazzein, a Sweet Protein, and the T1R2-T1R3 human Sweet receptor.
Journal of molecular biology, 2010Co-Authors: Fariba M. Assadi-porter, John L. Markley, Emeline Maillet, Jeniffer Quijada, James T. Radek, Marianna MaxAbstract:The Sweet Protein brazzein [recombinant Protein with sequence identical with the native Protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human Sweet receptor, a heterodimeric G-Protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the Sweet receptor):R217A/hT1R3 (human T1R3 subunit of the Sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the Sweet receptor by some mechanism other than the proposed wedge models.
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How Sweet It Is : Detailed Molecular and Functional Studies of Brazzein, a Sweet Protein and Its Analogs
2008Co-Authors: Fariba M. Assadi-porter, Marco Tonelli, James T. Radek, Claudia C. Cornilescu, John L. MarkleyAbstract:Brazzein is a small, low-calorie, Sweet Protein with high stability over wide temperature and pH ranges. Brazzein has desirable taste characteristics that resemble those of carbohydrate Sweeteners. Brazzein folds in a β-α-β 2 topology in which the α-helix packs against the three-stranded antiparallel β-sheet. This structure is held together by four disulfide bridges. We developed an efficient bacterial production system for brazzein that allows us to express wild-type and mutant Proteins. We have designed a large number of brazzein variants for taste tests. These include mutations that affect surface charges, disulfide bridges, loops, and flexible regions. We have subjected a subset of these variants to detailed analysis by NMR spectroscopy to identify patterns of hydrogen bonds and internal mobility. The results show a correlation between these physical properties and the Sweetness of the Protein. These results led us to propose a multi-site binding model for the interaction between brazzein and the heterodimeric human Sweet receptor, which we are continuing to test with the goal of designing more potent brazzein analogs as potential future Sweeteners.
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Efficient and rapid Protein expression and purification of small high disulfide containing Sweet Protein brazzein in E. coli
Protein expression and purification, 2007Co-Authors: Fariba M. Assadi-porter, Sammy Patry, John L. MarkleyAbstract:Brazzein Protein comes from an edible fruit, which has a long history of being a staple in the local human diet in Africa. The attractive features of brazzein as a potential commercial Sweetener include its small size (53 amino acid residues), its stability over wide ranges of temperature and pH, and the similarity of its Sweetness to sucrose. Heterologous production of brazzein is complicated by the fact that the Protein contains four disulfide bridges and requires a specific N-terminal sequence. Our previous protocol for producing the Protein from Escherichia coli involved several steps with low overall yield: expression as a fusion Protein, denaturation and renaturation, oxidation of the cysteines, and cleavage by cyanogen bromide at an engineered methionine adjacent to the desired N-terminus. The new protocol described here, which is much faster and leads to a higher yield of native Protein, involves the production of brazzein in E. coli as a fusion with SUMO. The isolated Protein product contains the brazzein domain folded with correct disulfide bonds formed and is then cleaved with a specific SUMO protease to liberate native brazzein. This protocol represents an important advancement that will enable more efficient research into the interaction between brazzein and the receptor as well as investigations to test the potential of brazzein as a commercially viable natural low calorie Sweetener.
