Role of hydrophobic amino acids in gurmarin,a sweetness-suppressing polypeptide

The sweetness-suppressing polypeptide gurmarin isolated from Gymnema sylvestre consists of 35 amino acid residues and contains three intramolecular disulfide bonds. Nuclear magnetic resonance analysis showed that the hydrophobic side chains of Tyr-13, Tyr-14, Trp-28, and Trp-29 in gurmarin are oriented outwardly. Together with the hydrophobic side chains of Leu-9, Ile-11, and Pro-12, they form a hydrophobic cluster, and therefore these hydrophobic groups are assumed to act as the site for interaction with the receptor protein. To examine the roles of these hydrophobic amino acids, they were replaced by Gly. The resulting [Gly^13,14,28,29]gurmarin and [Gly^{9,11,13,14,28,29}]gurmarin did not suppress the responses to sucrose, glucose, fructose, or Gly. This result strongly suggests that these hydrophobic amino acids are involved in the interaction with the receptor protein. © 1998 John Wiley & Sons, Inc. Biopoly 45: 231–238, 1998     

Evidence for a lack of functional receptors for nerve growth factor (NGF) in chick bone cells in vitro

Nerve growth factor (NGF) is essential for the development and differentiation of sympathetic and sensory neurons. Recently, NGF receptors were demonstrated in non-neural cells, and several mesenchymal cell types including lymphocytes and skeletal myotubes were shown to be stimulated to proliferate by NGF. Our purpose was to examine for the presence of functional NGF receptors in osteoblasts. Bone cells from chick calvaria were used as a model; PC-12 cells derived from rat adrenal pheochromocytoma were used as positive controls. NGF was examined for functions in chick bone cells by studying effects on (1) [³H]-thymidine incorporation; (2) alkaline phosphatase (ALP) activity; and (3) protein tyrosine phosphorylation. Effects of NGF on thymidine incorporation and protein tyrosine phosphorylation by PC-12 cells were also measured. A radioreceptor assay was used to test for the presence of receptors. In chick calvarial cells, NGF had no effect on thymidine incorporation, ALP activity or protein tyrosine phosphorylation. Radioreceptor assay with bone cells showed no evidence of NGF receptors. In contrast, in PC-12 cells, NGF (1) decreased thymidine incorporation; (2) increased protein tyrosine phosphorylation; and (3) showed receptor activity by radioreceptor assay. In conclusion, unlike several other mesenchymal cell types, chick bone cells show no evidence of NGF receptors or functional responses to NGF in vitro.     

Antigenicities of stem bromelain. Contribution of three-dimensional structure and individual amino acid residues

The contribution of three-dimensional structure and individual amino acid residues to the antigenicities of macromolecular protein was investigated for a thiol protease stem bromelain as antigen. The extent of the participation was demonstrated by a decrease in antigenicity when the enzyme was denatured in 8 M urea before and after reductive cleavage of intrapeptide disulfide bonds or modified in particular amino acid residues. The results showed that the enzyme treated with 8 M urea without reductive cleavage of disulfide bonds preserved about 90% of antigenicity to antibodies against native stem bromelain, while the enzyme denatured after the reductive cleavage of disulfide bonds brought about almost 80% disappearance of the antigenicity. Modification of individual amino acid side chains revealed that lysine was the most immunodominant amino acid, showing 2.5% contribution per residue, and tyrosine followed with 1.2%. However, acidic amino acids such as flutamic and aspartic acids were found to be as low as 0.3%, and tryptophan was 0.2%. These data suggest that most of the antigenic determinants of stem bromelain are of the steric conformation in which lysine and/or tyrosine are most frequently involved as immunodominant amino acids.     

红花菜豆(矮生红花变种)凝集素分子的色氨酸残基修饰与其活性的关系

用各种化学试剂修饰红花菜豆（Ｐｈａｓｅｏｌｕｓｃｏｃｃｉｎｅｕｓｖａｒｒｕｂｒｏｎａｎｕｓ,Ｂｅｒｒｙ）凝集素（简称ＰＣＬ）分子,测定与其活性相关的氨基酸残基．经ＮＢＳ修饰表明ＰＣＬ具有８个Ｔｒｐ残基,其中４个暴露于分子表面,此４个Ｔｒｐ残基被修饰后,ＰＣＬ的凝血活性完全丧失．比较ＰＣＬ修饰前后的ＣＤ光谱表明修饰不改变其二级结构。修饰Ｔｙｒ,Ａｒｇ,Ｈｉｓ残基和游离氨基及羧基不影响ＰＣＬ的血凝活性．巯基也不是血凝活性所必需,但是ＰＣＬ分子中的二硫键被还原,或被ＣＮＢｒ分解为两个片断则使蛋白质丧失血凝活性,提示分子的完整结构对ＰＣＬ的血凝活力是重要的     

On the Role of Disulfide Bonds in Polymerization of Soybean 7S Globulin during Storage

Modification of soybean 7S globulin with N-ethylmaleimide (NEM) was effective for preventing humidity-induced insolubilization during storage. The sulfhydryl-disulfide interchange reaction and/or oxidation of the sulfhydryl groups to form disulfide bonds closely related to the polymerization of the 7S globulin. A dimer, consisting of α′ and α subunits linked with disulfide bonds, was observed in the polymerized 7S globulin fractions, but this dimer was not readily apparent in the NEM-7S globulin. The formation of the dimer certainly initiates the insolubilization of the 7S globulin during storage. Although the β subunit had sulfhydryl groups, it did not take a part in the formation of the dimer.     

Extracellular production of lipoxygenase from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on wheat protein

In this study, the lipoxygenase (ana-LOX) gene from Anabaena sp. PCC 7120 was successful expressed and secreted in Bacillus subtilis. Under the control of the P43 promoter, with a signal peptide from the B. subtilis 168 nprB gene, and facilitated by the molecular chaperone PrsA, the production of the recombinant ana-LOX (ana-rLOX) reached 76 U/mL (171.9 μg/ml) in the supernatant. The purified ana-rLOX was investigated for its effect on dough protein. Ana-rLOX treatment decreased free sulfhydryl groups, increased glutenin macropolymer content, promoted the formation of covalent bonds between gluten protein, and affected protein crosslinking. The results indicated that large aggregates involving gliadin and glutenin were formed. The glutenin macropolymer played a role in the formation of the dough network structure through the exchange of thiol disulfide bonds and the formation of hydrogen or hydrophobic bonds with other proteins.     

What can disulfide bonds tell us about protein energetics, function and folding: simulations and bioninformatics analysis

We study the impact of disulfide bonds on protein stability and folding. Using lattice model simulations, we show that formation of a disulfide bond stabilizes a protein to an extent that depends on the distance along the chain between linked cysteine residues. However, the impact of disulfide bonds on folding kinetics varies broadly, from acceleration when disulfides are introduced in or close to the folding nucleus, to slowing when disulfides are introduced outside the nucleus. Having established the effect of disulfide bonds on stability, we study the correlation between the number of disulfide bonds and the composition of certain amino acid classes with the goal to use it as a statistical probe into factors that contribute to stability of proteins. We find that the number of disulfides is negatively correlated with aliphatic hydrophobic but not aromatic content. It is surprising that we observe a strong correlation of disulfide content with polar (Q,S,T,N) amino acid content and a strong negative correlation with charged (E,D,K,R) content. These findings provide insights into factors that determine protein stability and principles of protein design as well as possible relations of disulfide bonds and protein function.     

A novel lipid transfer protein from the dill Anethum graveolens L.: isolation,structure, heterologous expression,and functional characteristics

A novel lipid transfer protein, designated as Ag‐LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag‐LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag‐LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag‐LTP spatial structure contains four α ‐helices, forming the internal hydrophobic cavity, and a long C‐terminal tail. The measured volume of the Ag‐LTP hydrophobic cavity is equal to ~800 A³, which is much larger than those of other plant LTP1s. Ag‐LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

The essential cell division protein FtsN contains a critical disulfide bond in a non‐essential domain

Disulfide bonds are found in many proteins associated with the cell wall of Escherichia coli, and for some of these proteins the disulfide bond is critical to their stability and function. One protein found to contain a disulfide bond is the essential cell division protein FtsN, but the importance of this bond to the protein's structural integrity is unclear. While it evidently plays a role in the proper folding of the SPOR domain of FtsN, this domain is non‐essential, suggesting that the disulfide bond might also be dispensable. However, we find that FtsN mutants lacking cysteines give rise to filamentous growth. Furthermore, FtsN protein levels in strains expressing these mutants were significantly lower than in a strain expressing the wild‐type allele, as were FtsN levels in strains incapable of making disulfide bonds (dsb^‐) exposed to anaerobic conditions. These results strongly suggest that FtsN lacking a disulfide bond is unstable, thereby making this disulfide critical for function. We have previously found that dsb^‐ strains fail to grow anaerobically, and the results presented here suggest that this growth defect may be due in part to misfolded FtsN. Thus, proper cell division in E. coli is dependent upon disulfide bond formation.     

Influence of native disulfide bonds of globular proteins on their retention behavior on reverse phase supports

The reduction of the disulfide bonds of globular proteins, for example, those of lysozyme or ribonuclease-A, results in an increase in the hydrodynamic volume of the polypeptide chain. This is reflected in an earlier elution of the reduced protein on gel filtration compared to that of the native disulfide-bonded form. The reduction of the four disulfide bonds of ribonuclease-A increased its retention time on reverse phase support, suggesting an increase in the apparent hydrophobicity of the protein molecule on reduction. Performic acid-oxidized ribonuclease-A eluted ahead of native disulfide-bonded ribonuclease on RP HPLC, suggesting a decrease in the hydrophobicity of the molecule. However, the hydrodynamic volume of performic acid-oxidized ribonuclease-A is similar to that of reduced protein as reflected in its gel filtration behavior. Thus, the increased retention of the reduced protein compared to that of native disulfide-bonded protein is not related to the increased hydrodynamic volume, and is a reflection of the stronger interaction of reduced protein with the reverse phase support. Reoxidation of the reduced ribonuclease-A regenerated the original chromatographic behavior of the protein on the reverse phase support. Similar results were also obtained with hen egg white lysozyme. The results of the present study are interpreted as indicating that the native disulfide bonds of a globular protein restrict the exposure of the hydrophobic amino acid residues of the polypeptide chain with a consequent lower retention on the reverse phase support compared to its reduced form.     

Different dynamics and pathway of disulfide bonds reduction of two human defensins,a molecular dynamics simulation study

Human defensins are a class of antimicrobial peptides that are crucial components of the innate immune system. Both human α defensin type 5 (HD5) and human β defensin type 3 (hBD‐3) have 6 cysteine residues which form 3 pairs of disulfide bonds in oxidizing condition. Disulfide bond linking is important to the protein structure stabilization, and the disulfide bond linking and breaking order have been shown to influence protein function. In this project, microsecond long molecular dynamics simulations were performed to study the structure and dynamics of HD5 and hBD‐3 wildtype and analogs which have all 3 disulfide bonds released in reducing condition. The structure of hBD‐3 was found to be more dynamic and flexible than HD5, based on RMSD, RMSF, and radius of gyration calculations. The disulfide bridge breaking order of HD5 and hBD‐3 in reducing condition was predicted by two kinds of methods, which gave consistent results. It was found that the disulfide bonds breaking pathways for HD5 and hBD‐3 are very different. The breaking of disulfide bonds can influence the dimer interface by making the dimer structure less stable for both kinds of defensin. In order to understand the difference in dynamics and disulfide bond breaking pathway, hydrophilic and hydrophobic accessible surface areas (ASA), buried surface area between cysteine pairs, entropy of cysteine pairs, and internal energy were calculated. Comparing to the wildtype, hBD‐3 analog is more hydrophobic, while HD5 is more hydrophilic. For hBD‐3, the disulfide breaking is mainly entropy driven, while other factors such as the solvation effects may take the major role in controlling HD5 disulfide breaking pathway. Proteins 2017; 85:665–681. © 2016 Wiley Periodicals, Inc.     

The Antigenic Properties of β-Lactoglobulin Examined with Mouse IgE Antibody

The effects of enzymic or chemical fragmentations and of chemical modifications on the antigenic properties of bovine β-Mactoglobulin were examined using specific mouse IgE antibody. The antigenic reactivity of β-Mactoglobulin derivatives was represented in terms of their ability to neutralize specific IgE antibodies assayed by passive cutaneous anaphylaxis in rat. The tryptic, chymotryptic or peptic hydrolysate free of native β-Mactoglobulin had no antigenic reactivity but the fragments obtained after CNBr cleavage retained the ability to bind the antibody. Modification of the sulfhydryl group, arginine or tryptophan residues and amino groups had no effect on antigenic reactivity but a little decrease in the reactivity was observed on the cleavage of the two disulfide bridges. These results suggest that one sulfhydryl group, two arginine and tryptophan residues and most of the amino groups are out of antigenic sites in β-Mactoglobulin and that the antigenicity depends on the conformation maintained by the disulfide bridges.     

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG⁰) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.     

Hydrophobic fluorescent group coupled to Taka-amylase A. Change of its environmnt accompanying splitting of disulfide bonds

Native Taka-amylase A (α-amylase produced by Asp. oryzae) was coupled with 1-dimethylaminonaphthalene-5-sulfonyl chloride. When disulfide bonds of the modified enxyme were split by reduction or by reduction and subsequent carboxymethylation, its fluorescent properties changed markedly. It was suggested that the hydrophobic dye group is incorporated into a hydrophobic region as the consequence of the flexibility gained by the polypeptide chain by the cleavage of disulfide bonds.     

Amino acid sequence of a non-specific wheat phospholipid transfer protein and its conformation as revealed by infrared and Raman spectroscopy. Role of disulfide bridges and phospholipids in the stabilization of the alpha-helix structure.

A wheat non specific phospholipid transfer protein has been isolated from wheat seeds and its amino acid sequence reveals that it is composed of 90 residues for a molecular weight of 9607. From the comparison of its sequence with those of the eight known proteins of the same family, hypotheses on the role of some conserved residues in the transfer activity can be made. The conformation of this protein has been studied by Raman and Fourier transform infrared spectroscopy and this is the first report on the structure of non specific plant phospholipid transfer proteins. As opposed to previous studies on the structure prediction from the amino acid sequence, the results obtained show that plant non specific phospholipid transfer proteins are not almost entirely composed of beta-sheets. Instead, infrared results show that the wheat protein contains 41% alpha-helix and 19% beta-sheet structures, while 40% of the conformation is undefined or composed of turns. Raman spectroscopy shows that three disulfide bridges adopt a gauche-gauche-gauche conformation while the other exhibits a gauche-gauche-trans conformation, and that the two tyrosine residues are hydrogen bonded to water molecules. The cleavage of the disulfide bonds affects significantly the conformation of the protein, the extended confirmation being increased by 15% at the expense of the alpha-helix content. On the other hand, the binding of 1-palmitoyllysophosphatidylcholine to the protein leads to an increase of 8% of the alpha-helix content compared to the free protein. Secondary structure predictions from the amino acid sequence suggest that the binding of a phospholipid stabilizes helicity of the amphipathic helices while the reduction of disulfide bonds would affect the stability of the N-terminal helix. The extended structure located at the C-terminus is not affected. Finally, the wheat phospholipid transfer protein has no effect on the thermotropic behavior of large unilamellar vesicles of dimyristoylphosphatidylcholine while it increases the conformational order of the acyl chains of large unilamellar vesicles of dimyristoylphosphatidylglycerol in the liquid-crystalline state. No major conformational changes of the protein are observed when it is adsorbed to phospholipid vesicles. These results suggest that the helical structure is essential for the transfer activity without excluding a possible role of the C-terminal extended structure on the adsorption to phospholipid vesicles.     

Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.     

Construction of a synthetic protein using PCR with a high essential amino acid content for nutritional purposes

Ovalbumin is considered a protein of high nutritional value because it contains essential amino acids and is highly digestible. Therefore, it has a high biological value. Currently, the high food demand requires worldwide attention because food production is insufficient. Therefore, other alternatives are necessary to satisfy food demands, such as protein engineering. In this work, a protein with a high essential amino acid content similar to ovalbumin was synthesized by protein engineering, expressed, and digested in vitro. The assembly and sequential overlap extension PCR strategy was used to synthesize a 345-bp gene that encodes a high essential amino acid content protein (HEAAP). The 345-bp product was cloned into the vector pBAD TOPO®, and expressed in Escherichia coli BL21. PCR reactions and sequencing demonstrated the presence, orientation, and correct sequence of the insert. HEAAP expression was induced by l-arabinose and then purified using Ni–NTA affinity chromatography. The expression in E. coli was low and barely detected by Western blot assay. The in vitro multienzyme digestibility of HEAAP was around 79%, which suggests that the protein is potentially nutritious. Virtual analysis classifies the protein as unstable and hydrophilic, with a half-life in E. coli of 10 h. The recombinant HEAAP was successfully synthesized, but it is necessary to improve the digestibility and to optimize expression including selecting other expression models.

     

RNase activity requires formation of disulfide bonds and is regulated by the redox state

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitroby changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as -ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivoin wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.