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1.
The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Synthetic monellin is 4000 times as sweet as sucrose on a weight basis, and the native conformation is essential for the sweet taste. Knowledge of the active site of monellin will provide important information on the mode of interaction between sweeteners and their receptors. If the replacement of a certain amino acid residue in monellin removes the sweet taste, while the native conformation is retained, it may be concluded that the position replaced is the active site. Our previous replacement studies on Asp residues in the A chain did not remove the sweet taste. The B chain contains two Asp residues at positions 7 and 21, which were replaced by Asn. [AsnB21] Monellin and [AsnB7]monellin were 7000 and 20 times sweeter than sucrose, respectively. The low potency of the [AsnB7]monellin indicates that ASpB7 participates in binding with the receptor. ASpB7 was then replaced by Abu. [AbuB7]Monellin was devoid of sweetness, and retained the native conformation. ASpB7 is located at the surface of the molecule (Ogata et al.). These results suggest that Asp7 in the B chain is the highly probable active site of monellin.  相似文献   

2.
目的:合成单链莫内甜蛋白基因,构建其植物表达载体。方法与结果:根据已报道的单链莫内甜蛋白的氨基酸序列及甜味机理,重新设计合成了全长294bp的莫内甜蛋白基因,其中单链莫内甜蛋白氨基酸序列中的Asp69(原AspA16)突变为Asn。利用DNA重组技术,将莫内甜蛋白基因克隆到植物表达载体pBl221中,构建了莫内甜蛋白基因的植物表达载体pBI221-monellin。结论:构建了莫内甜蛋白基因的植物表达载体,为转化园艺植物以改善其口感奠定了基础。  相似文献   

3.
The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of these chains. The complete amino acid sequence of monellin was determined by a combination of FAB- and ESI-mass spectrometry, and by automatic Edman degradation.  相似文献   

4.
The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste. © 1998 John Wiley & Sons, Inc. Biopoly 46: 215–223, 1998  相似文献   

5.
Highly probable active site of the sweet protein monellin.   总被引:4,自引:0,他引:4  
The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Synthetic monellin is 4000 times as sweet as sucrose on a weight basis, and the native conformation is essential for the sweet taste. Knowledge of the active site of monellin will provide important information on the mode of interaction between sweeteners and their receptors. If the replacement of a certain amino acid residue in monellin removes the sweet taste, while the native conformation is retained, it may be concluded that the position replaced is the active site. Our previous replacement studies on Asp residues in the A chain did not remove the sweet taste. The B chain contains two Asp residues at positions 7 and 21, which were replaced by Asn. [AsnB21]Monellin and [AsnB7]monellin were 7000 and 20 times sweeter than sucrose, respectively. The low potency of the [AsnB7]monellin indicates that AspB7 participates in binding with the receptor. AspB7 was then replaced by Abu. [AbuB7]Monellin was devoid of sweetness, and retained the native conformation. AspB7 is located at the surface of the molecule (Ogata et al.). These results suggest that Asp7 in the B chain is the highly probable active site of monellin.  相似文献   

6.
Trikoningin KB II, a ten‐amino acid residue lipopeptaibol blocked at the N‐terminus by the n‐octanoyl group and at the C‐terminus by the 1,2‐amino alcohol l ‐leucinol, and extracted from the fungus Trichoderma koningii, exhibits membrane‐modifying properties. We have synthesized by solution‐phase methods trikoningin KB II and several analogues with acyl chains of different length at the N‐ and C‐termini. Permeability measurements showed that an appropriate length of the linear acyl chain is a more important characteristic for the onset of significant membrane‐modifying activity than its position in the peptide chain. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

7.
In an attempt to delineate the binding site(s) of monellin to the receptor by means of a structure-taste relationship, we synthesized four monellin analogues, [AsnA16]-, [AsnA22]-, [GlnA25]-, and [AsnA26]-monellin, which were 7500, 750, 2500, and 5500 times as sweet as sucrose on a weight basis, respectively. Among them, [AsnA22]monellin and [GlnA25]monellin were less sweet than monellin, and were susceptible to the HPLC conditions used. It can be concluded that Asp16, Asp22, Glu25, and Asp26 residues of the A chain did not participate in binding with the receptor, since the sweet taste was not removed by replacing the amino acid residues with Asn or Gln. It can also be concluded that Asp22 and Glu25 of the A chain may have participated in intramolecular binding, as was pointed out by Kim et al., since exchanging Asp22 and Glu25 of the A chain with Asn and Gln significantly decreased the stability in solution.  相似文献   

8.
甜蛋白Monellin基因在大肠杆菌中的高效表达   总被引:2,自引:0,他引:2  
据已报道的单链monellin甜蛋白的氨基酸序列,采用细菌偏爱密码子,人工合成了全长 294bp的 monellin基因。插入到大肠杆菌表达载体Pet_22b中,构建重组分泌型表达载体Petmo。经IPTG诱导Petmo所含有的甜蛋白基因可在大肠杆菌BL21(DE3)中高效表达,表达量占菌体可溶性蛋白的44.8%。且经纯化后测定其甜度是蔗糖的3000倍。得到的甜蛋白热稳性及耐酸性均比天然产物有所提高。  相似文献   

9.
Proteins are biosynthesized from N to C terminus before they depart from the ribosome and reach their bioactive state in the cell. At present, very little is known about the evolution of conformation and the free energy of the nascent protein with chain elongation. These parameters critically affect the extent of folding during ribosome‐assisted biosynthesis. Here, we address the impact of vectorial amino acid addition on the burial of nonpolar surface area and on the free energy of native‐like structure formation in the absence of the ribosomal machinery. We focus on computational predictions on proteins bearing the globin fold, which is known to encompass the 3/3, 2/2, and archaeal subclasses. We find that the burial of nonpolar surface increases progressively with chain elongation, leading to native‐like conformations upon addition of the last C‐terminal residues, corresponding to incorporation of the last two helices. Additionally, the predicted folding entropy for generating native‐like structures becomes less unfavorable at nearly complete chain lengths, suggesting a link between the late burial of nonpolar surface and water release. Finally, the predicted folding free energy takes a progressive favorable dip toward more negative values, as the chain gets longer. These results suggest that thermodynamic stabilization of the native structure of newly synthesized globins during translation in the cell is significantly enhanced as the chain elongates. This is especially true upon departure of the last C‐terminal residues from the ribosomal tunnel, which hosts ca., 30–40 amino acids. Hence, we propose that release from the ribosome is a crucial step in the life of single‐domain proteins in the cell. Proteins 2014; 82:2318–2331. © 2014 Wiley Periodicals, Inc.  相似文献   

10.
Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post‐synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C‐linked side chain are potentially useful building units for the synthesis of ferrocene‐containing peptides. We report here an efficient route to synthesize ferrocene‐containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2‐ferrocenyl‐1,3‐dithiane and iodides derived from aspartic acid or glutamic acid using n‐butyllithium leads to the incorporation of a ferrocenyl unit to the δ‐position or ε‐position of an α‐amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C‐terminus and N‐terminus of tripeptides in solution phase. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

11.
免疫性不育病人的血清(IPS)能100%地抑制人体外受精.用该血清筛选人睾丸cDNA基因表达文库,发现了一种新的睾丸特异抗原(称作C2).通过DNA顺序研究,并与基因库中的有关同源性基因数据进行比较,证实C2是一个新的特异蛋白.C2基因仅与睾丸组织的mRNA杂交.该克隆在大肠杆菌中所表达的融合蛋白能够被3个不同的不育病人血清识别.利用嵌套缺失和Western印迹的方法研究其抗原决定簇定位,发现B细胞抗原决定簇在羧基端29个氨基酸范围内.用GCG软件分析该抗原的氨基酸的亲水性和疏水性以及其存在于蛋白表面的可能性,确定其中的15个氨基酸为抗原决定簇所必需,并合成了多肽  相似文献   

12.
Biofouling, the undesirable accumulation of organisms onto surfaces, affects many areas including health, water, and energy. We previously designed a tripeptide that self‐assembles into a coating that prevents biofouling. The peptide comprises three amino acids: DOPA, which allows its adhesion to the surface, and two fluorinated phenylalanine residues that direct its self‐assembly into a coating and acquire it with antifouling properties. This short peptide has an ester group at its C‐terminus. To examine the importance of this end group for the self‐assembly and antifouling properties of the peptide, we synthesized and characterized tripeptides with different end groups (ester, amide, or carboxylic group). Our results indicate that different groups at the C‐terminus of the peptide can lead to a change in the peptide assembly on the surface and its adsorption process. However, this change only affects the antifouling properties of the coating toward Gram‐positive bacteria (Staphylococcus epidermidis), whereas Gram‐negative bacteria (Escherichia coli) are not affected.  相似文献   

13.
Transglutaminase (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins or peptides. Substrate specificities of TGase, Ca2+-independent microbial transglutaminase (MTGase), and Ca2+-dependent tissue type transglutaminase from guinea pig liver (GTGase) and fish, Red sea bream (Pagrus major), liver (FTGase), for acyl donors were investigated using synthetic peptides containing Gln residues and Gln analogues with different lengths of side chain. MTGase dose not recognize the Gln analogues as a substrate and has strict substrate specificities toward L-Gln. Substrate peptides with a variety of sequences around the Gln residue, GXXQXXG (X=G, A, S, L, V, F, Y, R, N, E, L) were synthesized and used as acyl donors. As an acyl acceptor, the fluorescent reagent monodancyl cadaverine was used and the reactions analyzed with RP-HPLC. Substitution of the C-terminal of a Gln residue with a hydrophobic amino acid accelerated the reaction by GTGase and FTGase. N-terminal substitution of Gln residues had similar effects on the reaction by MTGase.  相似文献   

14.
Transglutaminase (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins or peptides. Substrate specificities of TGase, Ca2+-independent microbial transglutaminase (MTGase), and Ca2+-dependent tissue type transglutaminase from guinea pig liver (GTGase) and fish, Red sea bream (Pagrus major), liver (FTGase), for acyl donors were investigated using synthetic peptides containing Gln residues and Gln analogues with different lengths of side chain. MTGase dose not recognize the Gln analogues as a substrate and has strict substrate specificities toward L-Gln. Substrate peptides with a variety of sequences around the Gln residue, GXXQXXG (X=G, A, S, L, V, F, Y, R, N, E, L) were synthesized and used as acyl donors. As an acyl acceptor, the fluorescent reagent monodancyl cadaverine was used and the reactions analyzed with RP-HPLC. Substitution of the C-terminal of a Gln residue with a hydrophobic amino acid accelerated the reaction by GTGase and FTGase. N-terminal substitution of Gln residues had similar effects on the reaction by MTGase.  相似文献   

15.
The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments. The reconstitution of monellin mutants containing large surface charge perturbations was compared to the thermostability of structurally equivalent single-chain monellin containing the same sets of mutations under varying salt concentrations. The affinity between monellin fragments is found to correlate with the thermostability of single chain monellin, indicating the involvement of the same underlying Coulombic interactions. This confirms that there are no principal differences in the interactions involved in folding and binding. Based on comparison with a previous mutational study involving hydrophobic core residues, the relative contribution of Coulombic interactions to stability and affinity is modest. However, the Coulombic perturbations only affect the association rates of reconstitution in contrast to perturbations involving hydrophobic residues, which affect primarily the dissociation rates. These results indicate that Coulombic interactions are likely to be of main importance for the association of protein assembly, relevant for functions of proteins.  相似文献   

16.
Site‐specific labeling of synthetic peptides carrying N‐methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N‐terminus was synthesized by the solid‐phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N‐terminus, leaving side chain amino group intact. The synthetic human β‐defensin‐2 carrying MeOGly at its N‐terminus or the side chain amino group of Lys10 reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N‐methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site‐specific labeling of linear synthetic peptides as well as disulfide‐containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

17.
The structure of monellin and its relation to the sweetness of the protein.   总被引:1,自引:0,他引:1  
The sweet protein monellin [1-3] has been shown to consist of two non-identical subunits of 50 and 42 amino acid residues, which were separated electrophoretically and chromatographically. Automatic sequential Edman degradation gave the complete sequence of the longer subunit, and a partial sequency of the shorter one. It was found that the sweetness of monellin requires the undissociated molecule. The individual subunits were not sweet, neither did they block the sweet sensation of sucrose or monellin. Blocking of the single SH of monellin abolished its sweetness as did reaction of the single methionyl residue with CNBr. Since the cysteinyl and methionyl residues appear to be adjacent, it is suggested that this part of the molecule is essential for its sweetness.  相似文献   

18.
Structural simplification and modification of natural products are always very important resources to antitumor drugs. By introducing various aminomethyl groups and amide groups into the phenanthrene ring of tylophorine, a novel series of tylophorine derivatives have been designed and synthesized, and their antiproliferative activities against MCF‐7, A549 and HepG‐2 cells have been evaluated, too. The results indicated that most of the prepared compounds exhibited good antitumor activities. Especially, one compound with an {ethyl[2‐(morpholin‐4‐yl)ethyl]amino}methyl group at the side chain exhibited the most significant cytotoxic effects.  相似文献   

19.
l-Aspartate alpha-decarboxylase (ADC), encoded by the panD gene, catalyzes the conversion of l-aspartate into beta-alanine. In the microorganisms, beta-alanine is required for the synthesis of pantothenate (vitamin B(5)), which is the precursor of 4'-phosphopantetheine and coenzyme A. We have determined the crystal structure of Helicobacter pylori ADC, a tetrameric enzyme, in two forms: the apo structure at 2.0 A resolution and the isoasparagine complex structure at 1.55 A resolution. All subunits of the tetramer are self-processed at the Gly24-Ser25 linkage, producing the smaller beta chain (residues 1-24) and the larger alpha chain (residues 25-117). Each subunit contains nine beta-strands and three alpha-helices; it is folded into the double-psi beta-barrel structure. In the apo structure, the new amino terminus of the alpha chain, Ser25, is converted into a pyruvoyl group. In the isoasparagine complex structure, the substrate analog is covalently attached to the pyruvoyl group. This structure represents the enzyme-substrate Schiff base intermediate that was proposed to form prior to the decarboxylation step in the catalytic cycle of ADC. Thus our study provides direct structural evidence for the reaction mechanism of ADC.  相似文献   

20.
Stereospecific introduction of a methyl group to the indole-3-side chain enhanced activity in our tryptamine-derived series of GnRH receptor antagonists. Further improvements were achieved by variation of the bicyclic amino moiety of the tertiary amide and by adjustment of the tether length to a pyridine or pyridone terminus. These modifications culminated in analogue 24, which had oral activity in a rat model and acceptable oral bioavailability and half-life in dogs and monkeys.  相似文献   

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