Sweetness Determinant Sites of Brazzein, a Small, Heat-Stable, Sweet-Tasting Protein

Assadi‐Porter, Fariba M.; Aceti, David J.; Markley, John L.

doi:10.1006/abbi.2000.1726

Cited by 90 publications

(175 citation statements)

References 19 publications

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“…Our previous mutagenesis studies suggested that two surface areas are involved in the sweetness of brazzein (14). Similar results had been observed with the other sweet proteins, thaumatin and monellin (46 -48).…”

Section: Discussionsupporting

confidence: 82%

“…The purification of brazzein from inclusion bodies followed our published protocol, which uses 6 M guanidinium chloride containing 10 mM EDTA and 100 mM dithiothreitol (13). Brazzein was refolded easily with regeneration of its four disulfide bridges; refolding and product purity were checked by reversed-phase high performance liquid chromatography (14). NMR samples consisted of 3-6 mg of pure lyophilized protein dissolved in 250 -500 l of 90% H 2 O and 10% D 2 O; the pH was adjusted to 5.2.…”

Section: Methodsmentioning

confidence: 99%

“…Four of the sites of mutation (Ala 2 insertion, H31A, R33A, and D50A) are spatially close to one another. Two of the mutants (Ala 2 insertion and H31A) have about twice the sweetness of wild-type brazzein; the other three mutants (R33A, R43A, and D50A) have greatly reduced sweetness (Table I) (14). R43A is essentially tasteless; it is only slightly sweet in powder form.…”

mentioning

confidence: 99%

“…Brazzein is small, stable, and easily investigated in solution by NMR spectroscopy. We earlier designed a synthetic gene coding for brazzein and developed an efficient bacterial production system (a fusion protein expressed in Escherichia coli) (13) and have used this approach to discover mutants with altered sweetness (14). This system also has allowed us to produce rapidly stable isotope-labeled brazzein variants for structural evaluation by NMR spectroscopy.…”

mentioning

confidence: 99%

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Correlation of the Sweetness of Variants of the Protein Brazzein with Patterns of Hydrogen Bonds Detected by NMR Spectroscopy

Assadi‐Porter

Abildgaard

Blad

et al. 2003

Journal of Biological Chemistry

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C atoms were collected from the six brazzein proteins labeled uniformly with carbon-13 and nitrogen-15. In wild-type brazzein, this approach identified 17 backbone hydrogen bonds. In the mutants, altered magnitudes of the couplings identified hydrogen bonds that were strengthened or weakened; missing couplings identified hydrogen bonds that were broken, and new couplings indicated the presence of new hydrogen bonds. Within the series of brazzein mutants investigated, a pattern was observed between sweetness and the integrity of particular hydrogen bonds. All three "sweet" variants exhibited the same pattern of hydrogen bonds, whereas all three "non-sweet" variants lacked one hydrogen bond at the middle of the ␣-helix, where it is kinked, and one hydrogen bond in the middle of ␤-strands II and III, where they are twisted. Two of the non-sweet variants lack the hydrogen bond connecting the N and C termini. These variants showed greater mobility in the Nand C-terminal regions than wild-type brazzein.

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Section: Discussionsupporting

confidence: 82%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Correlation of the Sweetness of Variants of the Protein Brazzein with Patterns of Hydrogen Bonds Detected by NMR Spectroscopy

Assadi‐Porter

Abildgaard

Blad

et al. 2003

Journal of Biological Chemistry

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“…Mutation of amino acid sequence can occur with or without effects on protein stability and function. Previous studies on mutations in brazzein have reported some effects on its sweet-tasting properties whereby some mutations have resulted in largest increase in sweetness, while other mutations have basically caused brazzein to be tasteless [1,[8][9][10][11]. The studies have examined the role of the surface charge of the molecule as being an important for brazzein's sweetness taste.…”

Section: Introductionmentioning

confidence: 99%

The Effect of Mutation in Brazzein Deduced from Mutational Sites and Sequence Carbon Content

Amri¹

2015

IJGG

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Abstract:Brazzein is a small sweet-tasting protein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon with potential of replacement of carbohydrate sweeteners. Carbon content analysis was used to examine the effect of mutation in brazzein's two regions at residues 29-33 and 39-43 with residue 36 reported to be important in sweet tasting of the protein. Analysis for local carbon density at the mutational sites for brazzein mutants with increased sweetness taste at residues 29 and 41 revealed normal carbon distribution curves with increased carbon frequency peak compared to the wild-type, consequently stabilized the local structure. Brazzein mutants with reduced sweetness taste at residue position 30, 33, 36 and 43 were mostly characterized by abnormal broadened distribution curve for carbon content with decreased frequency peak which destabilized the local structure and possibly leading to loss of protein functionality. Further analysis of carbon distribution profile along protein sequences of brazzein revealed a variation in carbon distribution between mutants with increased sweetness taste and those with decreased sweetness taste. Mutants with increased sweetness taste had carbon distribution profile balancing well conforming to the globular proteins which prefers to have 31.45% of carbon all along the sequence for stability. This study has provided further information and additional insights into protein atomic composition in brazzein and its role in understanding the effect of mutation.

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Expression of a triple mutational des‐pGlu brazzein in transgenic mouse milk

Lü

et al. 2022

FEBS Open Bio

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Brazzein has excellent potential for use as a sweetener because of its high level of sweetening potency and stability against extreme temperature and pH. It is extracted from the tropical and difficult—to‐cultivate African plant Pentadiplandra brazzeana , which hampers its commercial viability. Here we report the mammary‐specific expression of wildtype or triple mutational (H31R/E36D/E41A) des ‐pGlu brazzeins in the milk of transgenic mice. Using enzyme‐linked immunoassay (ELISA), western blot, and sweetness intensity testing, we confirmed that the triple mutation made the des ‐pGlu brazzein molecule 10,000 times sweeter than sucrose in a weight base, even after 10 min of incubation at 100 °C; in addition, the triple mutant was also significantly sweeter than the wildtype des ‐pGlu brazzein. This study provides new insights for producing brazzein or brazzein‐sweetened milk from animals for use in food and healthcare applications.

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Sweetness Determinant Sites of Brazzein, a Small, Heat-Stable, Sweet-Tasting Protein

Cited by 90 publications

References 19 publications

Correlation of the Sweetness of Variants of the Protein Brazzein with Patterns of Hydrogen Bonds Detected by NMR Spectroscopy

Correlation of the Sweetness of Variants of the Protein Brazzein with Patterns of Hydrogen Bonds Detected by NMR Spectroscopy

The Effect of Mutation in Brazzein Deduced from Mutational Sites and Sequence Carbon Content

Expression of a triple mutational des‐pGlu brazzein in transgenic mouse milk

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