Incorporation of beta-selenolo[3,2-b]pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography

beta-Selenolo[3,2-b]pyrrolyl-L-alanine that mimics tryptophan with the benzene ring of the indole moiety replaced by selenophene, was incorporated into human annexin V and barstar. This was achieved by fermentation and expression in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. The seleno- proteins were obtained in yields comparable to those of the wild-type proteins and exhibit full crystallographic isomorphism to the parent proteins, but expectedly show altered absorbance profiles and quenched tryptophan fluorescence. Since the occurrence of tryptophan residues in proteins is rare, incorporation of the electron-rich selenium-containing tryptophan surrogate into proteins represents a useful supplementation and even a promising novel alternative to selenomethionine for solving the phase problem in protein X-ray crystallography.     

Enzymatic syntheses of 6-(4H-selenolo[3,2-b]pyrrolyl)-L-alanine, 4-(6H-selenolo[2,3-b]pyrrolyl)-L-alanine, and 6-(4H-furo[3,2-b]pyrrolyl-L-alanine

6-(4H-Selenolo[3,2-b]pyrrolyl)-L-alanine 1, 4-(6H-selenolo[2,3-b]pyrrolyl)-L-alanine 2, and 6-(4H-furo[3,2-b]pyrrolyl)-L-alanine 3 have been synthesized via reactions of selenolo[3,2-b]pyrrole, selenolo[2,3-b]pyrrole, and furo[3,2-b]pyrrole, respectively, with L-serine. The reactions are catalyzed by Salmonella typhimurium tryptophan synthase.     

Synthesis and incorporation of [6,7]-selenatryptophan into dihydrofolate reductase

Until recently, the only selenium containing amino acid which could be used to completely substitute for a wild type amino acid was selenomethionine (SeMet). In the last decade the preparation of SeMet containing proteins has proved to be valuable tools in the determination of three-dimensional structure by multiwavelength anomalous diffraction (MAD) techniques. The potential utility of a selenium containing tryptophan analog, beta-seleno[3,2-b]pyrrolyl-L-alanine ([4,5]selenatryptophan), has recently been demonstrated in the literature. This finding shows promise for the bioincorporation of its positional isomer, beta-selenolo[2,3-b]pyrrolyl-L-alanine ([6,7]selenatryptophan), thereby adding to the essential arsenal of selenium-containing amino acids for use in the characterization of proteins. The synthesis of [6,7]selenatryptophan by enzymatic biotransformation with tryptophan synthase from selenolo[2,3-b]pyrrole was carried out as well as its characterization by NMR spectroscopy and thin layer chromatography. Selenatryptophyl dihydrofolate reductase ([6,7]SeTrp-DHFR) was then synthesized in vivo, purified, and found to exhibit no perturbations to enzymatic activity.     

Activation of a mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. Identification of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole and its reaction with DNA

A potent mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), isolated from a tryptophan pyrolysate, was activated metabolically by rat liver microsomes and bound to DNA. An active metabolite formed by rat liver microsomes was identified as 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2). Synthetic N-OH-Trp-P-2 reacted with DNA efficiently after O-acetylation or to a lesser extent under acidic conditions (pH 5.5), but did not react appreciably under neutral conditions. Acid hydrolysis of DNA modified by O-acetylated N-OH-Trp-P-2 (N-OAc-Trp-P-2) gave 3-(8-guanyl)amino-1-methyl-5H-pyrido[4,3-b]indole (Gua-Trp-P-2), which is the main modified base of DNA formed by Trp-P-2 in the presence of microsomes. The glycoside bond of the modified base was found to be cleaved by heating at 100° for 1 hr at pH 7.0. In this way, the modified base was liberated from DNA modified by N-OAc-Trp-P-2 in good yield. N-OAc-Trp-P-2 bound to guanyl cytidine more effectively than to guanylic acid, suggesting that covalent binding with guanyl moiety of DNA involves intercalation of the ultimate mutagen into a base pair.     

Determination of nonligand amino acids critical to [4Fe-4S]2+/+ assembly in ferredoxin maquettes.

The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which efficiently incorporates a single [4Fe-4S]2+/+ cluster with spectroscopic and electrochemical properties that are typical of natural bacterial ferredoxins. Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster. In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM-7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM. FdM-7 consists solely of the. CIACGAC. consensus ferredoxin core motif observed in natural protein sequences. Initially, all of the nonliganding amino acids were substituted for either glycine, FdM-7-PolyGly (.CGGCGGC.), or alanine, FdM-7-PolyAla (.CAACAAC.), on the basis of analysis of natural ferredoxin sequences. Both FdM-7-PolyGly and FdM-7-PolyAla incorporated little [4Fe-4S]2+/+ cluster, 6 and 7%, respectively. A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement either in the second or in the sixth core motif positions,.CIGCGGC. or.CGGCGIC., restored the iron-sulfur cluster binding capacity of the peptides to the level of FdM-7. Incorporation of an isoleucine into the fifth position,.CGGCIGC., which in natural ferredoxins is predominantly occupied by a glycine, resulted in a loss of [4Fe-4S] affinity. The substitution of leucine, tryptophan, and arginine into the second core motif position illustrated the stabilization of the [4Fe-4S] cluster by bulky hydrophobic amino acids. Furthermore, the incorporation of a single isoleucine into the second core motif position in a 16-amino acid ferredoxin maquette resulted in a 5-fold increase in the level of [4Fe-4S] cluster binding relative to that of the glycine variant. The protein design rules derived from this study are fully consistent with those derived from natural ferredoxin sequence analysis, suggesting they are applicable to both the de novo design and structure-based redesign of natural proteins.     

Chemical modifications of tryptophan residues in peptides and proteins

Chemical protein modifications facilitate the investigation of natural posttranslational protein modifications and allow the design of proteins with new functions. Proteins can be modified at a late stage on amino acid side chains by chemical methods. The indole moiety of tryptophan residues is an emerging target of such chemical modification strategies because of its unique reactivity and low abundance. This review provides an overview of the recently developed methods of tryptophan modification at the peptide and protein levels.     

Synthesis of new series of α-cyclodextrin esters as dopamine carrier molecule

A new series of amphiphilic α-cyclodextrins were synthesized by grafting N-acylated amino acids [valine, leucine, phenylalanine, methionine, and tryptophan (3a-e)] to the primary hydroxyl groups via ester bond formation. The synthetic pathway involves selective hexa-bromination of the primary hydroxyls followed by per-substitution with the carboxylate moiety of the N-acetyl residues in the presence of DBU (1,8-diazabicyclo[5,4,0]undec-7-ene). The ability of the synthetic compounds for the extraction of dopamine was studied. The results showed a considerable ability of some of the amphiphilic compounds for the extraction of dopamine into octanol phase from water. To complete the study, the binding affinity of dopamine toward the synthetic host molecules was calculated by using of the molecular docking technique.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain.

The incorporation of [3H]phenylalanine, [3H]tyrosine, and [3H]tryptophan into protein and amino acyl-tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl-tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl-tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl-tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both Km and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than Km.     

A study on the tricarboxylic acid cycle and the synthesis of acetylcholine in the lobster nerve

1. The biosynthetic origin of the amide substituent of N-(alpha-hydroxyethyl)lysergamide has been studied. 2. [1-(14)C]Acetate, [(14)C]formate, [2-(14)C]mevalonic acid lactone, [2-(14)C]indole, dl-[3-(14)C]tryptophan, dl-[3-(14)C]serine, dl-[2-(14)C]alanine and [2-(14)C]pyruvate were efficiently incorporated into the alkaloid, but not dl-[1-(14)C]alanine or [1-(14)C]pyruvate. 3. Only the dl-[2-(14)C]alanine- and [2-(14)C]pyruvate-derived alkaloid contained appreciable radioactivity in the amide substituent. 4. l-[(15)N]Alanine-derived alkaloid was shown to be specifically labelled in the amide nitrogen. However, l-[(14)C,(15)N]alanine was found to be incorporated into the methylcarbinolamide substituent with an appreciable increase in the (15)N/(14)C ratio, suggesting that alanine is not the direct precursor of this moiety.     

3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (trp-P-1) is incorporated into rat splenocytes,thymocytes, and hepatocytes through monoamine transporters and induces apoptosis

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which is a tryptophan pyrolysate formed during cooking, induces apoptosis in rat splenocytes, thymocytes, and hepatocytes. In this study, we investigated whether Trp-P-1 is transported into these cells and causes apoptosis. Trp-P-1 was immediately incorporated into rat splenocytes, thymocytes, and hepatocytes in a dose- and time-dependent manner. Dopamine and serotonin significantly competed with the uptake of Trp-P-1 into these cells, and nomifensine and indatraline, which are inhibitors of dopamine- and serotonin-transporters, respectively, markedly suppressed the uptake of Trp-P-1. On the other hand, amino acids including tryptophan did not compete with Trp-P-1. Inhibition of monoamine transporters using nomifensine and indatraline partially suppressed Trp-P-1-induced cell death in these cells. In hepatocytes, the inhibition of transporters prevented Trp-P-1-induced morphological changes and activation of caspase-3. These results demonstrated that Trp-P-1 is incorporated into the cells through monoamine transporters and induces apoptosis.     

Mass spectrometric determination of isotopically labeled tyrosines and tryptophans in photosynthetic reaction centers of Rhodobacter sphaeroides R-26

A synthetic medium for growing Rhodobacter sphaeroides R-26 is developed. This medium opened the way to the preparation of photosynthetic reaction centers incorporated with L-[4'-13C]tyrosine or L-[1'-15N]tryptophan. Gas chromatography combined with mass spectroscopy was used to estimate the metabolic incorporation of the labeled amino acid into the protein. Conditions were found for near-quantitative incorporation of labeled aromatic amino acids into the reaction center.     

Skeletal muscle protein and amino acid metabolism in hereditary mouse muscular dystrophy. Accelerated protein turnover and increased alanine and glutamine formation and release

Interactins between skeletal muscle protein and amino acid metabolism were investigated using C57BL and 129ReJ mice with hereditary muscular dystrophy. On incubation, hind limb muscle preparations from dystrophic mice released large quantities of amino acids, particularly alanine and glutamine which were increased 70% and 40% compared to muscles from carrier or control mice. The increased alanine release did not result from altered alanine oxidation to CO2 or reincorporation into protein. Alanine and glutamine formation from added amino acids were equal with dystrophic and control muscles. Incorporation in vitro of leucine, alanine, and glutamate into proteins of dystrophic muscle was 3- to 7-fold greater than control muscle, and the incorporation in vivo of [3H]- or [14C]arginine into muscle proteins was greater in extent and earlier in time with dystrophic as compared to control muscle. Proteins were also labeled in vivo using [guanido-14C]arginine. On incubation of these muscles in vitro, a 100% greater loss of label from protein was observed with dystrophic as compared to control preparations, and the appearance of label in the media was correspondingly increased. Sodium dodecyl sulfate-gel electrophoresis of dystrophic skeletal muscle showed numerous protein bands to be reduced in density, but autoradiographic studies demonstrated that these same bands were more highly labeled in vitro by [35S]methionine in dystrophic than in control muscle. Although insulin stimulation of glucose uptake was markedly blunted in dystrophic muscle, insulin inhibited alanine and glutamine release equally from both control and dystrophic muscle. These data indicate that alanine and glutamine formation and release are increased in hereditary mouse muscular dystrophy. An accelerated degradation and an increased resynthesis of many muscle proteins were also observed in dystrophic compared to control animals. This increased proteolysis may account for the increased alanine and glutamine formation in dystrophic muscle.     

Macromolecular Syntheses During Germination and Outgrowth of Bacillus subtilis Spores

Alanine and glucose used jointly are known to be necessary and sufficient for spore germination in Bacillus subtilis 168. By testing them separately, we have verified that alanine provokes optimal phase-darkening of the spores but inhibits macromolecular syntheses, while glucose is specifically needed for initiating those syntheses. By using them in succession we obtained evidence suggesting that: (i) sporal modifications which lead to phase-darkening must occur before macromolecular synthesis can start; (ii) the amino acid pool, on which the early protein synthesis is solely dependent, expands during incubation in alanine which allows degradative but prevents synthetic activities; and (iii) progression of degradations in alanine not promptly followed by syntheses in glucose produce a metabolic imbalance in the germinating spore. A sharp transition in the origin of building blocks was shown by using a tryptophan-defective mutant. At first the synthesis of proteins depended on pre-existing amino acids from turnover of sporal material since it occurred in the absence of any exogenous amino acid and its rate remained unaltered by supplying either all amino acids except tryptophan or tryptophan alone. Eventually, protein synthesis became dependent strictly on exogenous tryptophan and strongly on the supply of several other amino acids, not required later during vegetative growth. Clearly, by the start of outgrowth, all building blocks must be provided either by endogenous de novo synthesis or by exogenous supply.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain

The incorporation of [³H]phenylalanine, [³H]tyrosine, and [³H]tryptophan into protein and amino acyl–tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl–tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl–tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl–tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both K_m and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than K_m.     

Effect of glucocorticoides on the release of amino acids in the perfused rat hindquarter (author's transl)]

The release of amino acids by skeletal muscle was studied in the isolated perfused rat hindquarter. Adrenalectomy depressed the formation of glutamine and alanine as well as the efflux of all other amino acids measured. Betamethasone--a synthetic glucocorticoid--caused a significant increase in the efflux of nearly all amino acids up to the level of normal controls. The release of amino acids was also increased in perfused hindquarters of diabetic rats. On the other hand, insulin exhibited a depressing effect on the release of amino acids by hindquarters of normal rats. The metabolic integrity of the muscle tissue was proved by measuring creatine phosphate, ATP, ADP and water content as well as by the significant insulin effect on glucose uptake and on [14C]leucine incorporation into muscle proteins.     

Synthesis and antiviral evaluation of some novel tricyclic pyrazolo[3,4-b]indole nucleosides

Novel pyrazolo[3,4-b]indole nucleoside analogs were synthesized from the corresponding 3-formyl-2-chloroindole and 3-cyano-2-chloroindole nucleosides by treatment with hydrazine. Very few examples of pyrazolo[3,4-b]indole heterocycles have been published in the literature and this is the first synthesis of nucleoside analogs containing this heterocycle. These new pyrazolo[3,4-b]indole nucleosides were active against human cytomegalovirus and herpes simplex virus type 1, but this activity was not well separated from cytotoxicity.     

Isolation and Characterization of a Proteolipid in Defatted Soybean Meals

A proteolipid was isolated from the chloroform–methanol (2:1, by vol.) extract of defatted soybean meals by a modified Folch method. The proteolipid gave a yield of 0.05% of the defatted meals, and the ratio of protein and lipid was neary 3:4. The complex gave a single band containing both protein and lipid on polyacrylamide gel electrophoresis. TLC analysis of the lipid moiety showed that the major components were glycolipids and phospholipids. The protein moiety contained more hydrophobic amino acids and less acidic amino acids in comparison with the amino acid composition of soybean globulin. The protein moiety contained two kinds of protein component (I and II) which have molecular weights of 13,000 (I) and 15,000 (II) on SDS-urea polyacrylamide gel electrophoresis, and N-terminal amino acids of alanine (I) and glutamic acid (II). The apoprotein is a new protein and different from the whey proteins or globulins of soybean.     

The effects of electrical stimulation and ionic alterations on the metabolism of amino acids and proteins in excised superior cervical ganglia of the rat

Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas-liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA-insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA-insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0 , 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium-dependent process. The incorporation of ¹⁴C from [U-¹⁴C]glucose into glutamate (+ glutamine) and aspartate (+ asparagine) was greater in stimulated than in resting ganglia. However, the conversion of glutamate carbons from [U-¹⁴C]l -glutamate into aspartate was not affected by stimulation. Incorporation of ¹⁴C from [U-¹⁴C]glucose into glycine and serine was apparently not affected by stimulation during the 60 min of incubation. However, serine was the only amino acid which exhibited a higher specific radioactivity in stimulated ganglia than in resting ganglia incubated for 4 h in standard media. Lithium ions had the apparent specific effect of increasing the labelling with ¹⁴C from [U-¹⁴C]glucose into ornithine, and increasing the efflux and overall metabolism of serine in the ganglia. Incorporation of ¹⁴C from [U-¹⁴C]glucose into proteins was lower in the stimulated than in the resting ganglia if compensation was made for the higher radioactivity available in the total free amino acid pool of the stimulated ganglia. The rate of ¹⁴C incorporation from [U-¹⁴C]glutamate into the TCA-insoluble proteins of resting ganglia was greater when no other amino acids at concentrations approximating plasma levels were added to the bathing media; this rate was lower in stimulated than in resting ganglia.     

Light emission from tryptophan oxidation by hypobromous acid

The emission of ultraweak light from cells is a phenomenon associated with the oxidation of biomolecules by reactive oxygen species. The indole moiety present in tryptophan, serotonin and melatonin is frequently associated with the emission of light during the oxidation of these metabolites. This study presents results for hypobromous acid (HOBr) oxidation of tryptophan as a putative endogenous source of ultraweak light emission. We found that chemiluminescence elicited by the oxidation of tryptophan by HOBr was significantly higher than by hypochlorous acid (HOCl). This difference was related to secondary oxidation reactions, which were more intense using HOBr. The products identified during oxidation by HOCl, but depleted by using HOBr, were N‐formylkynurenine, kynurenine, 1,2,3,3a,8,8a‐hexahydro‐3a‐hydroxypyrrolo[2,3‐b]‐indole‐2‐carboxylic acid, oxindolylalanine and dioxindolylalanine. The emission of light is dependent on the free α‐amino group of tryptophan, and hence, the indole of serotonin and melatonin, although efficiently oxidized, did not produce chemiluminescence. The emission of light was even greater using taurine monobromamine and dibromamine as the oxidant compared to HOBr. A mechanism based on bromine radical intermediates is suggested for the higher efficiency in light emission. Altogether, the experimental evidence described in the present study indicates that the oxidation of free tryptophan or tryptophan residues in proteins is an important source of ultraweak cellular emission of light. This light emission is increased in the presence of taurine, an amino acid present in large amounts in leukocytes, where this putative source of ultraweak light emission is even more relevant. Copyright © 2012 John Wiley & Sons, Ltd.