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Sunghou Kim - One of the best experts on this subject based on the ideXlab platform.

  • Protein Engineering vol.2 no.8 pp.571-575, 1989 Redesigning a sweet protein: increased stability and renaturability
    2016
    Co-Authors: Sunghou Kim, Rosalind Kim, Chui-hee Kang, Joong Myung Cho, Yong-beom Lee, Tae-kyu Lee
    Abstract:

    'To whom correspondence should be addressed Monellin is one of two natural proteins from African berries with potent sweet taste. Monellin is the smaller of the two, and consists of two peptides. The protein loses sweetness when heated above 50°C under acidic pH. Based on the crystal structure of Monellin we have fused the two chains into a single chain using several different linkers copied and 'transplanted ' from the same molecule. One of the newly designed proteins is as potently sweet as the natural one, is more stable upon temperature or pH changes, and renatures easily even after heating to 100°C at low pH. Key words: heat stability/Monellin/protein engineering/sweet taste/protein sweetene

  • on the mechanism of chaperone activity of the small heat shock protein of methanococcus jannaschii
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Rosalind Kim, Kyeong Kyu Kim, Hisao Yokota, Luhua Lai, Hihong Lee, Gangwon Cheong, Susan Marqusee, Sunghou Kim
    Abstract:

    The small heat-shock protein (sHSP) from Methanococcus jannaschii (Mj HSP16.5) forms a homomeric complex of 24 subunits and has an overall structure of a multiwindowed hollow sphere with an external diameter of ≈120 A and an internal diameter of ≈65 A with six square “windows” of ≈17 A across and eight triangular windows of ≈30 A across. This sHSP has been known to protect other proteins from thermal denaturation. Using purified single-chain Monellin as a substrate and a series of methods such as protease digestion, antibody binding, and electron microscopy, we show that the substrates bind to Mj HSP16.5 at a high temperature (80°C) on the outside surface of the sphere and are prevented from forming insoluble substrate aggregates in vitro. Circular dichroism studies suggest that a very small, if any, conformational change occurs in sHSP even at 80°C, but substantial conformational changes of the substrate are required for complex formation at 80°C. Furthermore, deletion mutation studies of Mj HSP16.5 suggest that the N-terminal region of the protein has no structural role but may play an important kinetic role in the assembly of the sphere by “preassembly condensation” of multiple monomers before final assembly of the sphere.

  • small heat shock protein of methanococcus jannaschii a hyperthermophile
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Rosalind Kim, Kyeong Kyu Kim, Hisao Yokota, Sunghou Kim
    Abstract:

    Small heat shock proteins (sHSPs) belong to a family of 12- to 43-kDa proteins that are ubiquitous and are conserved in amino acid sequence among all organisms. A sHSP homologue of Methanococcus jannaschii, a hyperthermophilic Archaeon, forms a homogeneous multimer comprised of 24 monomers with a molecular mass of 400 kDa in contrast to other sHSPs that show heterogeneous oligomeric complexes. Electron microscopy analysis revealed a spherically shaped oligomeric structure ≈15–20 nm in diameter. The protein confers thermal protection of other proteins in vitro as found in other sHSPs. Escherichia coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100°C, whereas extracts from cells not expressing the protein were heat-sensitive at 60°C. Similar results were obtained when purified sHSP protein was added to an E. coli cell lysate. The protein also prevented the aggregation of two purified proteins: single-chain Monellin (SCM) at 80°C and citrate synthase at 40°C.

  • structure of Monellin refined to 2 3 a resolution in the orthorhombic crystal form
    Acta Crystallographica Section D-biological Crystallography, 1997
    Co-Authors: G Bujacz, Craig M Ogata, Sunghou Kim, Maria Miller, Robert W Harrison, N Thanki, Gary L Gilliland, Alexander Wlodawer
    Abstract:

    The structure of orthorhombic crystals of Monellin, a sweet protein extracted from African serendipity berries, has been solved by molecular replacement and refined to 2.3 A resolution. The final R factor was 0.150 for a model with excellent geometry. A Monellin molecule consists of two peptides that are non-covalently bound, with chain A composed of three β-strands interconnected by loop regions and chain B composed of two β-strands interconnected by an α-helix. The N terminus of chain A is in close proximity to the C terminus of chain B. The two molecules in the asymmetric unit are related by a non-crystallographic twofold axis and form a dimer, similar to those previously observed in other crystal forms of both natural and single-chain Monellin. The r.m.s, deviation between the Cα atoms in the two independent molecules is 0.60 A, while the deviations from the individual molecules in the previously reported monoclinic crystals are 0.50–0.57 A. This result proves that the structure of Monellin is not significantly influenced by crystal packing forces.

Delia Picone - One of the best experts on this subject based on the ideXlab platform.

  • High-level production of single chain Monellin mutants with enhanced sweetness and stability in tobacco chloroplasts
    Planta, 2018
    Co-Authors: Daniela Castiglia, Serena Leone, Delia Picone, Rachele Tamburino, Lorenza Sannino, Jole Fonderico, Chiara Melchiorre, Andrea Carpentieri, Stefania Grillo, Nunzia Scotti
    Abstract:

    Main conclusion Plastid-based MNEI protein mutants retain the structure, stability and sweetness of their bacterial counterparts, confirming the attractiveness of the plastid transformation technology for high-yield production of recombinant proteins. The prevalence of obesity and diabetes has dramatically increased the industrial demand for the development and use of alternatives to sugar and traditional sweeteners. Sweet proteins, such as MNEI, a single chain derivative of Monellin, are the most promising candidates for industrial applications. In this work, we describe the use of tobacco chloroplasts as a stable plant expression platform to produce three MNEI protein mutants with improved taste profile and stability. All plant-based proteins were correctly expressed in tobacco chloroplasts, purified and subjected to in-depth chemical and sensory analyses. Recombinant MNEI mutants showed a protein yield ranging from 5% to more than 50% of total soluble proteins, which, to date, represents the highest accumulation level of MNEI mutants in plants. Comparative analyses demonstrated the high similarity, in terms of structure, stability and function, of the proteins produced in plant chloroplasts and bacteria. The high yield and the extreme sweetness perceived for the plant-derived proteins prove that plastid transformation technology is a safe, stable and cost-effective production platform for low-calorie sweeteners, with an estimated production of up to 25–30 mg of pure protein/plant.

  • Sweeter and stronger: enhancing sweetness and stability of the single chain Monellin MNEI through molecular design
    Scientific Reports, 2016
    Co-Authors: Serena Leone, Piero Andrea Temussi, Andrea Pica, Antonello Merlino, Filomena Sannino, Delia Picone
    Abstract:

    Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, Monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction.

  • molecular dynamics driven design of ph stabilized mutants of mnei a sweet protein
    PLOS ONE, 2016
    Co-Authors: Serena Leone, Delia Picone
    Abstract:

    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.

  • design of sweet protein based sweeteners hints from structure function relationships
    Food Chemistry, 2015
    Co-Authors: Michele F Rega, Serena Leone, Rossella Di Monaco, Silvana Cavella, Federica Donnarumma, Roberta Spadaccini, Delia Picone
    Abstract:

    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.

Piero Andrea Temussi - One of the best experts on this subject based on the ideXlab platform.

  • Sweeter and stronger: enhancing sweetness and stability of the single chain Monellin MNEI through molecular design
    Scientific Reports, 2016
    Co-Authors: Serena Leone, Piero Andrea Temussi, Andrea Pica, Antonello Merlino, Filomena Sannino, Delia Picone
    Abstract:

    Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, Monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction.

  • a hypersweet protein removal of the specific negative charge at asp21 enhances thaumatin sweetness
    Scientific Reports, 2016
    Co-Authors: Tetsuya Masuda, Piero Andrea Temussi, Keisuke Ohta, Naoko Ojiro, Kazuki Murata, Bunzo Mikami, Fumito Tani, Naofumi Kitabatake
    Abstract:

    Thaumatin is an intensely sweet-tasting protein that elicits sweet taste at a concentration of 50 nM, a value 100,000 times larger than that of sucrose on a molar basis. Here we attempted to produce a protein with enhanced sweetness by removing negative charges on the interacting side of thaumatin with the taste receptor. We obtained a D21N mutant which, with a threshold value 31 nM is much sweeter than wild type thaumatin and, together with the Y65R mutant of single chain Monellin, one of the two sweetest proteins known so far. The complex model between the T1R2-T1R3 sweet receptor and thaumatin, derived from tethered docking in the framework of the wedge model, confirmed that each of the positively charged residues critical for sweetness is close to a receptor residue of opposite charge to yield optimal electrostatic interaction. Furthermore, the distance between D21 and its possible counterpart D433 (located on the T1R2 protomer of the receptor) is safely large to avoid electrostatic repulsion but, at the same time, amenable to a closer approach if D21 is mutated into the corresponding asparagine. These findings clearly confirm the importance of electrostatic potentials in the interaction of thaumatin with the sweet receptor.

  • solution structure of a sweet protein nmr study of mnei a single chain Monellin
    Journal of Molecular Biology, 2001
    Co-Authors: Roberta Spadaccini, Orlando Crescenzi, Teodorico Tancredi, Natashia De Casamassimi, Gabriella Saviano, Roberta Scognamiglio, Alberto Di Donato, Piero Andrea Temussi
    Abstract:

    Abstract The sweet protein MNEI is a construct of 96 amino acid residues engineered by linking, with a Gly-Phe dipeptide, chains B and A of Monellin, a sweet protein isolated from Discoreophyllum cuminsii. Here, the solution structure of MNEI was determined on the basis of 1169 nuclear Overhauser enhancement derived distance restraints and 184 dihedral angle restraints obtained from direct measurement of three-bond spin coupling constants. The identification of hydrogen bonded NH groups was obtained by a combination of H/2H exchange data and NH resonance temperature coefficients derived from a series of HSQC spectra in the temperature range 278–328 K. The good resolution of the structure is reflected by the Z-score of the quality checking program in WHAT IF (−0.61). The topology of MNEI, like that of natural Monellin and of SCM, another single-chain Monellin, is typical of the cystatin superfamily: an α-helix cradled into the concave side of a five-strand anti-parallel β-sheet. The high resolution (14 restraints/residue) 3D structure of MNEI shows close similarity to the crystal structures of natural Monellin and of SCM but differs from the solution structure of SCM. The structures of SCM in the crystal and in solution differ in some of the secondary structure elements, but most of all in the relative arrangement of the elements: the four main β-strands that surround the helix in the crystal structure of SCM, are displaced far from the helix in the solution structure of SCM. These differences were attributed to the fact that SCM is a monomer in solution and a dimer in the crystal. This result is at variance with the observation that our solution structure, like that of SCM, corresponds to a monomeric state of the protein, as demonstrated by the insensitivity of HSQC spectra to extreme dilution (down to 20 μM). On the basis of the solution structure of MNEI it is possible to propose that the main glucophores are hosted on loop L34, whereas the N-terminal and C-terminal regions host two other important interaction regions, centered around segments 6–9 and 94–96.

Graeme L Conn - One of the best experts on this subject based on the ideXlab platform.

  • Reduced sweetness of a Monellin (MNEI) mutant results from increased protein flexibility and disruption of a distant poly-(L-proline) II helix
    Chemical Senses, 2011
    Co-Authors: Catherine M. Templeton, Jeanette R. Hobbs, Saeideh Ostovar Pour, Ewan W Blanch, Steven D. Munger, Graeme L Conn
    Abstract:

    Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain Monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding.

  • Chem. Senses doi:10.1093/chemse/bjr007 Reduced Sweetness of a Monellin (MNEI) Mutant Results from Increased Protein Flexibility and Disruption of a Distant Poly-(L-Proline) II Helix
    2011
    Co-Authors: Catherine M. Templeton, Jeanette R. Hobbs, Saeideh Ostovar Pour, Ewan W Blanch, Steven D. Munger, Graeme L Conn
    Abstract:

    Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain Monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding

Göran Hellekant - One of the best experts on this subject based on the ideXlab platform.

  • Monkey Electrophysiological and Human Psychophysical Responses to Mutants of the Sweet Protein Brazzein: Delineating Brazzein Sweetness
    2015
    Co-Authors: Zheyuan Jin, Fariba M. Assadi-porter, John L. Markley, Göran Hellekant
    Abstract:

    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

  • 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, 2005
    Co-Authors: Qin Zhao, Vicktoria Danilova, Göran Hellekant, Zheyuan Jin, Jikui Song, John L. Markley
    Abstract:

    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).

  • Brazzein a Small, Sweet Protein: Discovery and Physiological Overview
    Chemical Senses, 2005
    Co-Authors: Göran Hellekant, Vicktoria Danilova
    Abstract:

    , 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

  • Monkey Electrophysiological and Human Psychophysical Responses to Mutants of the Sweet Protein Brazzein: Delineating Brazzein Sweetness
    Chemical senses, 2003
    Co-Authors: Zheyuan Jin, Vicktoria Danilova, Fariba M. Assadi-porter, John L. Markley, Göran Hellekant
    Abstract:

    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.

  • Solution structure of the thermostable sweet-tasting protein brazzein.
    Nature structural biology, 1998
    Co-Authors: Jane E. Caldwell, Göran Hellekant, Frits Abildgaard, Željko Džakula, Ding Ming, J L Markley
    Abstract:

    The fruit of Pentadiplandra brazzeana Baillon contains a small, sweet-tasting protein named brazzein. The structure of brazzein in solution was determined by proton nuclear magnetic resonance spectroscopy at pH 5.2 and 22 degrees C. The brazzein fold, which contains one alpha-helix and three strands of antiparallel beta-sheet, does not resemble that of either of the other two sweet-tasting proteins with known structures, Monellin and thaumatin. Instead, the structure of brazzein resembles those of plant gamma-thionins and defensins and arthropod toxins. Sequence comparisons predict that members of a newly-identified family of serine proteinase inhibitors share the brazzein fold.