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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, Vicktoria Danilova, 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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Structural role of the terminal disulfide bond in the sweetness of Brazzein.
Chemical senses, 2011Co-Authors: Sannali M. Dittli, John L. Markley, Marco Tonelli, Marianna Max, Hongyu Rao, Jeniffer Quijada, Fariba M. Assadi-porterAbstract:Brazzein, a 54 residue sweet-tasting protein, is thought to participate in a multipoint binding interaction with the sweet taste receptor. Proposed sites for interaction with the receptor include 2 surface loops and the disulfide bond that connects the N- and C-termini. However, the importance of each site is not well understood. To characterize the structural role of the termini in the sweetness of Brazzein, the position of the disulfide bond connecting the N- and C-termini was shifted by substituting K3-C4-K5 with C3-K4-R5. The apparent affinity and Vmax of the C3-K4-R5-Brazzein (CKR-Brazzein) variant were only modestly decreased compared with the wild-type (WT) Brazzein. We determined a high-resolution structure of CKR-Brazzein by nuclear magnetic resonance spectroscopy (backbone root mean square deviation of 0.39 A ˚ ). Comparing the structure of CKR-Brazzein with that of WT-Brazzein revealed that the terminal b-strands of the variant display extended b-structure and increased dynamics relative to WT-Brazzein. These results support previous mutagenesis studies and further suggest that, whereas interactions involving the termini are necessary for full function of Brazzein, the termini do not constitute the primary site of interaction between Brazzein and the sweet taste 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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Direct NMR Detection of the Binding of Functional Ligands to the Human Sweet Receptor, a Heterodimeric Family 3 GPCR
Journal of the American Chemical Society, 2008Co-Authors: Fariba M. Assadi-porter, Marco Tonelli, Emeline Maillet, Klaas Hallenga, Outhiriaradjou Benard, Marianna Max, John L. MarkleyAbstract:We present a robust method for monitoring the binding of ligands to the heterodimeric (T1R2+T1R3) human sweet receptor (a family 3 GPCR receptor). The approach utilizes saturation transfer difference (STD) NMR spectroscopy with receptor proteins expressed on the surface of human epithelial kidney cells. The preparation investigated by NMR can contain either live cells or membranes isolated from these cells containing the receptor. We have used this approach to confirm the noncompetitive binding of alitame and cyclamate to the receptor and to determine that greatly reduced receptor binding affinity compared to wild-type Brazzein explains the lack of sweetness of Brazzein mutant A16C17. This approach opens new avenues for research on the mechanism of action of the sweet receptor and for the design of new noncalorigenic sweeteners.
Kwang-hoon Kong - One of the best experts on this subject based on the ideXlab platform.
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Efficient Brazzein production in yeast (Kluyveromyces lactis) using a chemically defined medium
Bioprocess and Biosystems Engineering, 2021Co-Authors: Se-woong Park, Byung-ha Kang, Hyeong-min Lee, Sung-jun Lee, Han-seul Kim, Hye-won Choi, Tae Jung Park, Kwang-hoon KongAbstract:The sweet-tasting protein Brazzein offers considerable potential as a functional sweetener with antioxidant, anti-inflammatory, and anti-allergic properties. Here, we optimized a chemically defined medium to produce secretory recombinant Brazzein in Kluyveromyces lactis , with applications in mass production. Compositions of defined media were investigated for two phases of fermentation: the first phase for cell growth, and the second for maximum Brazzein secretory production. Secretory Brazzein expressed in the optimized defined medium exhibited higher purity than in the complex medium; purification was by ultrafiltration using a molecular weight cutoff, yielding approximately 107 mg L^−1. Moreover, the total media cost in this defined medium system was approximately 11% of that in the optimized complex medium to generate equal amounts of Brazzein. Therefore, the K. lactis expression system is useful for mass-producing recombinant Brazzein with high purity and yield at low production cost and indicates a promising potential for applications in the food industry.
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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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Optimized production and quantification of the tryptophan-deficient sweet-tasting protein Brazzein in Kluyveromyces lactis.
Preparative biochemistry & biotechnology, 2019Co-Authors: Hyung-min Lee, Sung-jun Lee, Se-woong Park, Kwang-hoon KongAbstract:The sweet-tasting protein Brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize Brazzein as a sweetener, optimization of fermentation an...
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Antioxidant, anti-inflammatory, and anti-allergic activities of the sweet-tasting protein Brazzein.
Food chemistry, 2017Co-Authors: Ju-hee Chung, Ji-na Kong, Hyo-eun Choi, Kwang-hoon KongAbstract:Sweet-tasting proteins may be useful as low-calorie sugar substitutes in foods, beverages, and medicines. Brazzein is an attractive sweetener because of its high sweetness, sugar-like taste, and good stability at high temperature and wide pH ranges. To investigate the bioactivities of Brazzein, the antibacterial, antifungal, antioxidant, anti-inflammatory, and anti-allergic activities were determined in vitro. Brazzein showed no antibacterial and antifungal activities, although it showed approximately 45% or greater similarity to defensin, which has antimicrobial effects, and drosomycin, which is used as an antifungal agent. However, Brazzein exhibited strong antioxidant effects, showing ABTS radical scavenging activity (IC50=12.55μM) and DPPH activity (IC50>30μM). Brazzein also showed anti-inflammatory activity and anti-allergic activity in a β-hexosaminidase assay (IC50>15μM) and cyclooxygenase-2 inhibition assay (IC50=12.62μM), respectively. These results suggest that Brazzein has antioxidant, anti-inflammatory, and anti-allergic activities and considerable potential as a functional sweetener.
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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 e...
Loïc Briand - One of the best experts on this subject based on the ideXlab platform.
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Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris
Life (Basel Switzerland), 2021Co-Authors: Fabrice Neiers, Christine Belloir, Nicolas Poirier, Christian Naumer, Michael Krohn, Loïc BriandAbstract:Brazzein is a small sweet-tasting protein found in the red berries of a West African evergreen shrub, Pentadiplandra brazzeana Baillon. Brazzein is highly soluble and stable over a large pH range and at high temperatures, which are characteristics that suggest its use as a natural sweetener. However, Pentadiplandra brazzeana culture is difficult at a large scale, limiting the natural source of Brazzein. Heterologous expression of Brazzein has been established in numerous systems, including bacteria, yeast, and transgenic plants. Brazzein requires four disulfide bonds to be active in eliciting an intense sweet taste, and the yeast Pichia pastoris appears to be one of the best options for obtaining functional Brazzein in high quantities. Employing yeast secretion in the culture medium allows us to obtain fully active Brazzein and facilitate purification later. To increase yeast secretion, we compared seven different signal peptides to successfully achieve Brazzein secretion using the yeast P. pastoris. The Brazzein proteins corresponding to these signal peptides elicited activation of the sweet taste receptor functionally expressed in a cellular assay. Among these tested signal peptides, three resulted in the secretion of Brazzein at high levels.
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Structure-function relationships of Brazzein, a sweet-tasting protein and its interactions with the human sweet taste receptor
2018Co-Authors: Fabrice Neiers, Loïc BriandAbstract:Brazzein is a small heat- and pH-stable sweet-tasting protein isolated from the West African plant, Pentadiplandra brazzeana. Brazzein combines a highly sweet potency, a long history of human consumption, and a remarkable stability, giving it great potential as a natural sweetener. Due to the difficulties of obtaining Brazzein from its natural source, several efforts have been made to express Brazzein using various heterologous expression systems. Brazzein like all classes of sweet compounds (natural sugars, natural and artificial sweeteners) are perceived through the activation of the T1R2/T1R3 heterodimeric sweet taste receptor. T1R2 and T1R3 subunits are members of the small family of class C G-protein coupled receptors (GPCRs). Class C GPCRs possess a large N-terminal domain (NTD) linked to a heptahelical transmembrane domain by a cysteine rich domain (CRD). Cellular assays, molecular docking and site-directed mutagenesis studies have revealed that the NTD of T1R2 (T1R2-NTD) contain the primary binding site for most of the sweet ligands including natural sugars and, artificial and natural sweeteners including sweet-tasting proteins. We will summarize the structure-activity relationship of Brazzein and will describe the current knowledge on the putative molecular mechanism of receptor activation, which remains to be elucidated.
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Development of Brazzein variants as sweet taste enhancers
2018Co-Authors: Loïc Briand, Fabrice NeiersAbstract:Development of Brazzein variants as sweet taste enhancers. Annual Meeting Innovation Alliance NatLifE 2020
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Development of Brazzein mutants with sweetness-enhancing
2016Co-Authors: Loïc BriandAbstract:Development of Brazzein mutants with sweetness-enhancing. Annual Meeting Innovation Alliance NatLifE 2020
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The Recent Development of a Sweet-Tasting Brazzein and its Potential Industrial Applications
2016Co-Authors: Fabrice Neiers, Naumer Christian, Krohn Michael, Loïc BriandAbstract:Brazzein is a small heat- and pH-stable sweet-tasting protein isolated from the West African plant, Pentadiplandra brazzeana. Brazzein combines a highly sweet potency, a long history of human consumption, and a remarkable stability, giving it great potential as a natural sweetener. Due to the difficulties of obtaining Brazzein from its natural source, several efforts have been made to express Brazzein using various heterologous expression systems. This chapter describes the biochemical, structural, sensory, and physiological properties of Brazzein. We will summarize the current knowledge of the structure-activity relationship of Brazzein. The biotechnological production of Brazzein using various expression systems will also be reviewed. Furthermore, the emerging application of Brazzein in the food industry to replace traditional sugars by acting as a natural, good, low-calorie sweetener will be discussed.
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, Vicktoria Danilova, 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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A Method to Measure Taste Qualities, Taste Intensity, and Temporal Profile of Compounds Aimed at Human Consumption by Taste Nerve Recordings in Monkeys
Sweetness and Sweeteners, 2008Co-Authors: Göran Hellekant, Yiwen WangAbstract:A method is described to measure taste qualities and intensity of compounds aimed at human consumption by recording from taste nerve fibers of monkeys. Here we demonstrate its usefulness by presenting results of a comparison of sweetness of Brazzein derivatives and bitterness of denatonium benzoate analogs as assessed by a human taste panel and recorded from monkey single taste fibers. The correlation between the responses in sweet sensitive fibers in monkeys and the estimates of sweetness by a human taste panel was 0.78 for 25 analogs of the sweet protein Brazzein, and 0.9 between the responses of bitter sensitive fibers and human bitterness estimates for 6 analogs of the bitter compound denatonium benzoate.
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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, Göran Hellekant, Zheyuan Jin, Vicktoria Danilova, 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).
Masaru Tanokura - One of the best experts on this subject based on the ideXlab platform.
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Brazzein and structurally similar proteins: structural/functional comparisons
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Koji Nagata, Nobuko Hongo, Yasuhiro Kameda, Akihiro Yamamura, Hiroshi Sasaki, Woo Cheol Lee, Kohki Ishikawa, Ei-ichiro Suzuki, Masaru TanokuraAbstract:Brazzein, a 6.5-kDa protein consisting of 54 amino acids and four disulfide bonds, is the smallest sweet-tasting protein yet isolated from the wild African plant Pentadiplandra brazzeana. Brazzein has various desirable properties for use as a low-calorie sweetener in the diets of individuals suffering from diabetes, obesity, and metabolic syndrome. For example, Brazzein has a high water solubility and a high thermostability. In addition, Brazzein is 2000-times sweeter than sucrose on a weight basis. Both the solution and crystal structures of Brazzein have been reported. In the crystal structure [1], Brazzein has a defensin-like fold containing two α-helices and a three-stranded antiparallel β-sheet. Defensins are small cysteine-rich cationic proteins found in both animals and plants, which function by binding to the microbial cell membrane, and, once embedded, forming pore-like membrane defects that allow efflux of essential ions and nutrients. In fact, Yount and Yeaman reported that Brazzein has antimicrobial activity against Gram positive (Bacillus subtilis and Staphylococcus aureus) and negative (Escherichia coli) bacteria and a fungus (Candida albicans) at pH 7.5 rather than pH 5.5 [2]. A search for proteins with a similar backbone fold to Brazzein using the DALI server shows that structurally similar proteins to Brazzein include plant defensins, scorpion neurotoxins (K+ channel blockers), arthropod defensins, mollusc defensins, mold defensins, and a plant trypsin inhibitor. These proteins commonly have a γ-core sequence. Here we compare their sequences, structures and functions, which has led to a conclusion that the C-terminal half of Brazzein is important for its antimicrobial activity, Brazzein will not have a neurotoxin activity, and it will not act as a trypsin inhibitor.
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The structure of Brazzein, a sweet‐tasting protein from the wild African plant Pentadiplandra brazzeana
Acta Crystallographica Section D Biological Crystallography, 2013Co-Authors: Koji Nagata, Nobuko Hongo, Yasuhiro Kameda, Akihiro Yamamura, Hiroshi Sasaki, Woo Cheol Lee, Kohki Ishikawa, Ei-ichiro Suzuki, Masaru TanokuraAbstract:Brazzein is the smallest sweet-tasting protein and was isolated from the wild African plant Pentadiplandra brazzeana. The Brazzein molecule consists of 54 amino-acid residues and four disulfide bonds. Here, the first crystal structure of Brazzein is reported at 1.8 A resolution and is compared with previously reported solution structures. Despite the overall structural similarity, there are several remarkable differences between the crystal and solution structures both in their backbone folds and side-chain conformations. Firstly, there is an additional α-helix in the crystal structure. Secondly, the atomic r.m.s.d.s between the corresponding Cα-atom pairs are as large as 2.0–2.2 A between the crystal and solution structures. Thirdly, the crystal structure exhibits a molecular shape that is similar but not identical to the solution structures. The crystal structure of Brazzein reported here will provide additional information and further insights into the intermolecular interaction of Brazzein with the sweet-taste receptor.
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The structure of Brazzein, a sweet-tasting protein from the wild African plant Pentadiplandra brazzeana.
Acta crystallographica. Section D Biological crystallography, 2013Co-Authors: Koji Nagata, Nobuko Hongo, Yasuhiro Kameda, Akihiro Yamamura, Hiroshi Sasaki, Woo Cheol Lee, Kohki Ishikawa, Ei-ichiro Suzuki, Masaru TanokuraAbstract:Brazzein is the smallest sweet-tasting protein and was isolated from the wild African plant Pentadiplandra brazzeana. The Brazzein molecule consists of 54 amino-acid residues and four disulfide bonds. Here, the first crystal structure of Brazzein is reported at 1.8 Å resolution and is compared with previously reported solution structures. Despite the overall structural similarity, there are several remarkable differences between the crystal and solution structures both in their backbone folds and side-chain conformations. Firstly, there is an additional α-helix in the crystal structure. Secondly, the atomic r.m.s.d.s between the corresponding C(α)-atom pairs are as large as 2.0-2.2 Å between the crystal and solution structures. Thirdly, the crystal structure exhibits a molecular shape that is similar but not identical to the solution structures. The crystal structure of Brazzein reported here will provide additional information and further insights into the intermolecular interaction of Brazzein with the sweet-taste receptor.
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Crystallization and preliminary X‐ray analysis of Brazzein, a new sweet protein
Acta Crystallographica Section D Biological Crystallography, 1996Co-Authors: Kohki Ishikawa, Masaru Tanokura, M. Ota, Y. Ariyoshi, H. Sasaki, D. Ming, J. Caldwell, F. AbilgaadAbstract:Brazzein is a sweet protein isolated from a wild African plant Pentadiplandra brazzeana. Brazzein is the smallest (molecular mass = 6473 Da) and the most water-soluble protein sweetener discovered so far and is highly thermostable. Crystals were grown by vapor diffusion using sodium sulfate as a precipitant. They belong to the tetragonal space group I4(1)22 with unit-cell parameters a = b = 61.4, c = 59.6 A and with one molecule in the asymmetric unit. The crystals diffract to 1.8 A resolution using synchrotron radiation.