Absence Of Trp Research Articles

Development of Amphiphilic Short Peptides to Control Drug Inclusivity through Metal Complexation Reactions

We previously reported that amphiphilic peptides containing one Lys and two His in the hydrophilic domain—KHHAAAAAA7 (KHHA7)—formed ternary complexes with intermediate metal ions and a poorly water-soluble anticancer drug—paclitaxel (Ptx)—which improved the water dispersibility of Ptx. In this study, the effect of ternary complexes of KHHA7 on cell viability is evaluated in vitro using HeLa cells. The ternary complexes reduced the cell viability by approximately 60%, suggesting that complex formation enhanced the cytotoxicity by increasing the water dispersibility of Ptx. Furthermore, the novel amphiphilic short peptides KHHAAAAAAW (KHHA6W) and KHHAAAWAAA (KHHA3WA3) were synthesized by Fmoc solid phase synthesis as dispersants for Ptx. These peptides differed from KHHA7 owing to the presence or absence of Trp and its position. The change in amino acid residues from Ala to Trp and the position of Trp in the hydrophobic domain of the amphiphilic peptide had little effect on the water dispersibility of Ptx.

Ultrasensitive and reusable electrochemical aptasensor for detection of tryptophan using of [Fe(bpy)3](p-CH3C6H4SO2)2 as an electroactive indicator

In this paper, we report the application of a reusable electrochemical aptasensor for detection of tryptophan by using [Fe(bpy)3](p-CH3C6H4SO2)2 as an electroactive indicator and based on the target-compelled aptamer displacement. The aptasensor fabricated by self-assembling the thiolated probe on the surface of graphite screen-printed electrode modified with gold nanoparticles/multiwalled carbon nanotubes and chitosan nanocomposite (AuNPs/MWCNTs-Chit/SPE). Afterward, Trp aptamer (Apt) immobilized on the modified electrode surface through hybridization. In the absence of Trp, a sharp peak of [Fe(bpy)3](p-CH3C6H4 SO2)2 can be observed in differential pulse voltammetry (DPV) study. The introduction of Trp led to the formation of aptamer-Trp complex and dissociation of the aptamer from the DNA-Apt duplex on the electrode surface into the solution and decreases the peak current intensity of electroactive indicator. This is because, [Fe(bpy)3](p-CH3C6H4SO2)2 tends to bind to the two strands DNA. Therefore, the peak current of [Fe(bpy)3](p-CH3C6H4 SO2)2 linearly decreased with increasing the concentration of Trp over a range of 3.0 nM- 100 μM. The detection limit (3 σ) was 1.0 nM. In addition, we examined the selectivity of the constructed biosensor for tyrosine, histidine, arginine, lysine, valine and methionine that belonged to the amino acid family.The obtained results showed that the fabricated sensor had a good selectivity for Trp against the other examined amino acids. Also, the potential applicability of the aptasensor was investigated by detecting the Trp in a complex media such as human blood plasma spiked with Trp.

Biophysical insight into the binding of triprolidine hydrochloride to human serum albumin: Calorimetric, spectroscopy and molecular docking approaches

Triprolidine hydrochloride is extensively used as antihistamine and anticholinergic drug. We have examine the binding mechanism of triprolidine hydrochloride (TRP) with human serum albumin (HSA) using fluorescence spectroscopy, circular dichroism (CD), isothermal titration calorimetry (ITC) and molecular docking techniques. The fluorescence data demonstrated that TRP binds to HSA through static quenching. The thermodynamic parameters (∆H, ∆S and ∆G), binding stoichiometry and the binding constant between TRP and HSA were determined using ITC. Protein surface hydrophobicity of HSA has been calculated in presence and absence of TRP. CD results suggest the possibility of variation in the conformation of HSA in the presence of TRP. The binding site I was confirmed by molecular docking technique. The esterase-like activity of HSA shows that Arg-410 and Tyr-411 of sub-domain IIIA were directly involved in the binding process. Chemical unfolding study of HSA was carried out in the presence of TRP using GuHCl by CD and fluorescence spectroscopy.

Tryptophan depletion under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through reactive oxygen species-dependent and independent pathways.

In atherosclerosis-associated pathologic entities characterized by malnutrition and inflammation, L-tryptophan (TRP) levels are low. Insulin resistance is an independent cardiovascular risk factor and induces endothelial dysfunction by increasing fatty acid oxidation. It is also associated with inflammation and low TRP levels. Low TRP levels have been related to worse cardiovascular outcome. This study evaluated the effect of TRP depletion on endothelial dysfunction under conditions that imitate insulin resistance. Fatty acid oxidation, harmful pathways due to increased fatty acid oxidation, and endothelial dysfunction were assessed in primary human aortic endothelial cells cultured under normal glucose, low insulin conditions in the presence or absence of TRP. TRP depletion activated general control non-derepressible 2 kinase and inhibited aryl hydrocarbon receptor. It increased fatty acid oxidation by increasing expression and activity of carnitine palmitoyltransferase 1. Elevated fatty acid oxidation increased the formation of reactive oxygen species (ROS) triggering the polyol and hexosamine pathways, and enhancing protein kinase C activity and methylglyoxal production. TRP absence inhibited nitric oxide synthase activity in a ROS-dependent way, whereas it increased the expression of ICAM-1 and VCAM-1 in a ROS independent and possibly p53-dependent manner. Thus, TRP depletion, an amino acid whose low levels have been related to worse cardiovascular outcome and to inflammatory atherosclerosis-associated pathologic entities, under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through ROS-dependent and independent pathways. These findings may offer new insights at the molecular mechanisms involved in accelerated atherosclerosis that frequently accompanies malnutrition and inflammation.

UVA illumination-induced optical coupling between tryptophan and natural dissolved organic matter.

Exposure of tryptophan (Trp) in aqueous solutions to UVA radiation resulted in decrease of Trp (C1) but generated an unknown fluorescent component (C2) with fluorescence emission maxima extending into wavelength range characteristic of humic substance (HS)-like material. The intensity of the two components (C1 and C2) could be operationally fit to linear functions of the illumination time t (0~40 h). However, C1 and C2 decreased and increased nonlinearly respectively in a mixture (Trp mixed with a reference sample of natural organic matter, i.e., NOM) which was exposed to the same UVA illumination, and the change of both C1 and C2 was faster than that in the absence of NOM. Moreover, the UV-Vis absorption maximum (ex = 278 nm) of Trp was faster removed for the mixture (after 5 h) than for Trp solutions without NOM (after 20 h). These observations suggested NOM-facilitated photobleaching of Trp and photoproduction of a new FDOM component under UVA illumination. Meanwhile, the fluorescence of the NOM in the absence of Trp was well represented by two HS-like components which decayed monotonically upon exposure to UVA light, while the photoinduced decay became nonmonotonic in the presence of Trp, and one component even increased with illumination during certain time window, indicating Trp-facilitated production of HS-like fluorescence signatures from NOM. The findings show that UVA-induced optical signature changes of tryptophan and HS-like materials are coupled and highlight the potential impact of absorption of solar UVA light by natural dissolved organic matter (DOM) on using the optical signatures to trace sources and sinks of DOM.

Kinetic Analysis of Correct Nucleotide Insertion by a Y-family DNA Polymerase Reveals Conformational Changes Both Prior to and following Phosphodiester Bond Formation as Detected by Tryptophan Fluorescence

The Sulfolobus solfataricus Y-family DNA polymerase Dpo4 is a model for translesion replication and has been used in the analysis of individual steps involved in catalysis. The role of conformational changes has not been clear. Introduction of Trp residues into the Trp-devoid wild-type protein provided fluorescence probes of these events, particularly in the case of mutants T239W and N188W. With both mutants, a rapid increase in Trp fluorescence was observed only in the case of normal base pairing (G:C), was saturable with respect to dCTP concentration, and occurred in the absence of phosphodiester bond formation. A subsequent decrease in the Trp fluorescence occurred when phosphodiester bond formation was permitted, and these rates were independent of the dCTP concentration. This step is relatively slow and is attributed to a conformational relaxation step occurring after pyrophosphate release, which was measured and shown to be fast in a separate experiment. The measured rate of release of DNA from Dpo4 was rapid and is not rate-limiting. Overall, the measurements provide a kinetic scheme for Dpo4 different than generally accepted for replicative polymerases or proposed for Dpo4 and other Y-family polymerases: the initial enzyme.DNA.dNTP complex undergoes a rapid (18 s(-1)), reversible (21 s(-1)) conformational change, followed by relatively rapid phosphodiester bond formation (11 s(-1)) and then fast release of pyrophosphate, followed by a rate-limiting relaxation of the active conformation (2 s(-1)) and then rapid DNA release, yielding an overall steady-state kcat of <1 s(-1).

An eGFP-Based Genetic Screen for Defects in Light-Triggered Subcelluar Translocation of the Drosophila Photoreceptor Channel TRPL

Signaling at the plasma membrane is modulated by up- and down-regulation of signaling proteins. A prominent example for this type of regulation is the Drosophila TRPL ion channel that changes its spatial distribution within the photoreceptor cell. In dark-raised flies TRPL is localized in the rhabdomeral photoreceptor membrane and it translocates to the cell body upon illumination. It has been shown that TRPL translocation depends on the activation of the phototransduction cascade and requires the presence of functional rhodopsin as well as Ca2+-influx through a second light-activated ion channel, TRP. However, little is known about the cell biological mechanism underlying TRPL translocation. Here we describe a FRT/FLP screen designed to isolate mutants defective in TRPL internalization based on the localization of eGFP-tagged TRPL in the eyes of living flies. We mutated chromosome arms 2L, 2R, and 3R and isolated 12 mutants that failed to internalize TRPL. We found that four mutants did not complement genes known to affect TRPL translocation, which are trp, ninaE, and inaD. Two of the isolated mutants represent new alleles of trp and ninaE. The trp allele contains a premature stop codon after amino acid 884, whereas the ninaE allele has a mutation resulting in the substitution P193S. As determined biochemically no TRP or rhodopsin protein, respectively, was expressed in the eyes of these mutants. The absence of TRP or rhodopsin in the isolated mutants readily explains the defect in TRPL internalization and proves the feasibility of our genetic screen.

The acute tryptophan depletion and loading tests: Specificity issues

Abstract The acute tryptophan (Trp) depletion (ATD) and loading tests are a powerful tool for examining the role of serotonin in normal subjects and in those with behavioral and other disorders. The question of how specific to serotonin the tests are, has, however, received little attention. In particular, two questions arise regarding the potential effects of the tests on catecholamine synthesis, which may confound interpretation of behavioral data, namely whether: (1) the absence of Trp or its addition alters the [Phe + Tyr]/[LNAA + Trp] ratio, thereby influencing the entry of tyrosine and phenylalanine into the brain; (2) the relatively larger amounts of the 3 large neutral amino acids (LNAA), namely Val, Leu and Ile, in the amino acid formulation, compared with those of Phe and Tyr, could actually decrease the above ratio. Furthermore, the [Phe + Tyr]/[LNAA + Trp] and/or the [Trp]/[CAA] ratio may also be altered in control balanced and/or in Trp-loaded amino acid formulations, given the disproportionate contents of these 6 competitors in the different formulations. We addressed these questions in 114 normal US subjects divided into 5 groups of 20–25 each receiving Trp-depleted, -loaded or -balanced amino acid formulations at two dose levels: 50 g and the traditional 100 g. As expected, the [Trp]/[CAA] ratio was decreased by ATD and increased after Trp loading. However, the [Phe + Tyr]/[LNAA + Trp] ratio was also decreased by both ATD and Trp loading at both dose levels of each formulation. In subjects receiving a 50 g balanced control amino acid formulation, both the [Trp]/[CAA] and the [Phe + Tyr]/[LNAA + Trp] ratios were decreased. In all cases, the above unwanted decreases were due to the relatively larger contents in the various formulations of the above 3 LNAA, compared with Phe and Tyr. Based on these results and on theoretical considerations, we suggest that by either decreasing the contents of the LNAA by up to ∼ 30% or increasing those of Phe and Tyr by up to ∼ 50%, the [Phe + Tyr]/[LNAA + Trp] ratio can be maintained at normal baseline levels after consumption of the depletion, loading or balanced formulation and the [Trp]/[CAA] ratio can also be kept unaltered in the latter control formulation. We believe that this approach could achieve a greater specificity for these Trp manipulations and thus enhance the validity of the ATD and loading tests and improve interpretation of behavioral and related data.

Production of indole-3-acetic acid, aromatic amino acid aminotransferase activities and plant growth promotion by Pantoea agglomerans rhizosphere isolates

The production of auxins, such as indole-3-acetic acid (IAA), by rhizobacteria has been associated with plant growth promotion, especially root initiation and elongation. Six indole-producing bacteria isolated from the rhizosphere of legumes grown in Saskatchewan soils and identified as Pantoea agglomerans spp. were examined for their ability to promote the growth of canola, lentil and pea under gnotobiotic conditions and for tryptophan (Trp)-dependent IAA production. Five of the isolates enhanced root length, root weight or shoot weight by 15–37% in at least one of the plant species, but isolates 3–117 and 5–51 were most consistent in enhancing plant growth across the three species. Indole concentrations in the rhizosphere of plants grown under gnotobiotic conditions increased in the presence of the rhizosphere isolates and when Trp was added 3 days prior to plant harvest. Isolates 3–117, 5–51 and 5–105 were most effective in increasing rhizosphere indole concentrations. Colony hybridization confirmed that all of the isolates possessed the ipdC gene which codes for a key enzyme in the Trp-dependent IAA synthetic pathway. The activity of amino acid aminotransferase (AAT), catalyzing the first step in the Trp-dependent synthetic pathway, was examined in the presence of Trp and other aromatic amino acids. All of the isolates accumulated Trp internally and released different amounts of IAA. The production of IAA from the isolates was greatest in the presence of Trp, ranging from 2.78 to 16.34 μg mg protein−1 in the presence of 250 μg of Trp ml−1. The specific activity of AAT was correlated with the concentration of IAA produced in the presence of Trp but not when tyrosine (Tyr), phenylalanine (Phe) or aspartate (Asp) was used as a sole nitrogen source. Isolate 3–117, which produced significant concentrations of IAA in the presence and absence of Trp, was able to use aromatic amino acids as sole sources of nitrogen and was most consistent in enhancing the growth of canola, lentil and pea may have potential for development as a plant growth-promoting inoculant.

Multiple open reading frames, codon bias and the evolution of the genetic code

Examination of over 1.08 million genes in the SWISS-PROT/TrEMBL data bank revealed that 6.8% of them have full length in-frame sense/antisense double open reading frames (SASDORFs) and 1.5% have full length triple open reading frames (TORFs). The frequency of occurrence of SASDORFs and TORFs in a million random composition genes is 0.14% and less than 0.001% respectively. Multiple open reading frames (MORFs) are associated with proteins that are ancient, essential, and present in most species. There is an extreme nucleotide triple bias in SASDORFs in many protein families including the short chain oxidoreductase (SCOR) enzymes, heat shock proteins, ABC transporters, and ribosomal proteins. SCOR enzyme genes exhibit a GC-rich triple bias similar to that of GC-rich bacteria, suggesting that the primordial SCOR enzyme evolved in a GC-rich species. Of particular interest is the high incidence of MORFs and the absence of Trp and Cys residues in ribosomal proteins (Rbps). Analysis of the sequences from many species of proteins having 30% homology with each of 14 bacterial 50S Rbps reveals that over 60% of them contain no Trp, 52% are missing Cys and 34% are missing both Cys and Trp. These findings suggest that MORFs, severe codon bias and the absence of Trp and Cys residues are not confined to GC-rich species and are hallmarks of ancient enzymes the encoding of which has not been altered by millions of years of evolution or lateral gene transfer. Supported in part by NIH Grant DK26546.

Structures of Tryptophanyl-tRNA Synthetase II from Deinococcus radiodurans Bound to ATP and Tryptophan: INSIGHT INTO SUBUNIT COOPERATIVITY AND DOMAIN MOTIONS LINKED TO CATALYSIS

An auxiliary tryptophanyl tRNA synthetase (drTrpRS II) that interacts with nitric-oxide synthase in the radiation-resistant bacterium Deinococcus radiodurans charges tRNA with tryptophan and 4-nitrotryptophan, a specific nitration product of nitric-oxide synthase. Crystal structures of drTrpRS II, empty of ligands or bound to either Trp or ATP, reveal that drTrpRS II has an overall structure similar to standard bacterial TrpRSs but undergoes smaller amplitude motions of the helical tRNA anti-codon binding (TAB) domain on binding substrates. TAB domain loop conformations that more closely resemble those of human TrpRS than those of Bacillus stearothermophilus TrpRS (bsTrpRS) indicate different modes of tRNA recognition by subclasses of bacterial TrpRSs. A compact state of drTrpRS II binds ATP, from which only minimal TAB domain movement is necessary to bring nucleotide in contact with Trp. However, the signature KMSKS loop of class I synthetases does not completely engage the ATP phosphates, and the adenine ring is not well ordered in the absence of Trp. Thus, progression of the KMSKS loop to a high energy conformation that stages acyl-adenylation requires binding of both substrates. In an asymmetric drTrpRS II dimer, the closed subunit binds ATP, whereas the open subunit binds Trp. A crystallographically symmetric dimer binds no ligands. Half-site reactivity for Trp binding is confirmed by thermodynamic measurements and explained by an asymmetric shift of the dimer interface toward the occupied active site. Upon Trp binding, Asp68 propagates structural changes between subunits by switching its hydrogen bonding partner from dimer interface residue Tyr139 to active site residue Arg30. Since TrpRS IIs are resistant to inhibitors of standard TrpRSs, and pathogens contain drTrpRS II homologs, the structure of drTrpRS II provides a framework for the design of potentially useful antibiotics.

Auxin production is a common feature of most pathovars of Pseudomonas syringae.

We investigated indole-3-acetic acid (IAA) production by 57 pathovars of Pseudomonas syringae and related species. Most of those analyzed produced IAA, especially in the presence of tryptophan. Eight strains produced high IAA concentrations in the absence of Trp. The iaaM and iaaH genes of P. savastanoi pv. savastanoi were detected in a limited number of strains only, including the eight above-mentioned strains. Thus, IAA synthesis in most assayed strains of P. syringae and related species does not involve genes highly similar to iaaM and iaaH. In contrast, the iaaL gene encoding an IAA-lysine synthase was detected in most pathovars, and was often found on plasmids.

Effect of dietary 5-hydroxy-L-tryptophan for prevention of scoliosis in tryptophan-deficient chum salmon fry.

Tryptophan (Trp)-deficient diets (Trp 50mg/100g diet) supplemented with graded levels of 5-hydroxy-L-tryptophan (5-HTP; 0.1, 1, 10 & 100mg/100g diet) were fed to chum salmon fry Oncorhynchus keta to clarify whether 5-HTP, the precursor of serotonin (5-HT), prevents scoliosis caused by Trp deficiency and affects the levels of neurotransmitters in fish brain. The administration of the dose of less than 10mg of 5-HTP/100g diet caused scoliosis in 62-65% of fish atfer 4 weekrearing, whereas the increase in dietary level of 100mg reduced this value to 1.7%. 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) contents in the brain of the fish fed Trp-deficient diet containing 100mg 5-HTP/100g diet as well as those of Trp-sufficient diet (Trp 290mg/100g diet) were higher than those of the other dietary groups. The brain norepinephrine (NE) contents showed a tendency to rise as the supplemental level of 5-HTP increased. These results clearly indicate that the oral administration of 5-HTP up to the level of 100mg/100g dict or 40mg/kg body weight/day can almost completely prevent occurrence of scoliosis. Incidence of scoliosis in the fish fed Trp-free diet (Trp 0%) was only 15% even though brain 5-HT content of this group was in the same range as those fed Trp-deficient diet containing supplemental 5-HTP up to 10mg/100g diet. However, the brain NE and dopamine contents of the former were almost the same as those fed Trp-sufficient diet. These findings suggest that catecholamine may be also involved in an occurrence of scoliosis and that there is a considerable physiological difference between complete absence of Trp and intake of small quantity of Trp.

Control of RNA and protein synthesis by the concentration of Trp-tRNA Trp in Escherichia coli infected with bacteriophage MS2

The effect of varying the concentration of Trp-tRNATrp in Escherichia coli infected with bacteriophage MS2 has been studied by varying the amount of exogenously added tryptophan (Trp) to cells bearing a mutation which results in a tryptophanyl-tRNA synthetase with a higher Km value for Trp. The phenotype of the mutant has been confirmed by measuring the level of tRNATrp which can be aminoacylated in vivo, and the mutant has also been shown to have elevated tRNATrp levels compared to wild-type. The growth of MS2 decreases continuously with decreasing Trp concentration (and hence, decreasing Trp-tRNATrp concentration). This appears to be due to reduced gene expression, since at later times in infection the amount of MS2 coat protein synthesized likewise decreases continuously with decreasing Trp concentration. However, there is little decrease in the amount of coat protein or replicase synthesized during the first few minutes after the Trp concentration shift. A continuous increase in the average polysome size distribution is seen as the Trp concentration is decreased. MS2 RNA synthesis also decreases continuously with decreasing Trp concentration, and is shut off in the absence of Trp. This does not seem to be due to ppGpp as these cells are functionally relaxed under these conditions, nor does it seem to be due to degradation of pre-existing template. Addition of chloramphenicol abolishes the effect of Trp concentration on RNA synthesis. The data are consistent with a model in which ribosomes pause at Trp codons in the absence of Trp-tRNATrp, while other ribosomes queue behind and continue to load onto the message. The reduction of RNA synthesis would then be a consequence of coupling to translation.

A model for the tryptophan mediated tailfiber extension of bacteriophage T6

The T-even bacteriophages (T2, T4 and T6) have 6 long fibers that are attached to the end of the tail. The fibers may be in a retracted conformation, folded back along the tail, or in an extended conformation, in which case adsorption to host bacteria becomes possible. In some strains of T4 and T6 the aminoacid L-tryptophan (Trp) is required for fiber extension. We studied the reaction of phage T6 with Trp in 50 mM phosphate buffer pH 7.0 by measurements of transient electric birefringence (which gives the rotational diffusion coefficient D ), sedimentation coefficient and biological activity. For ~ particles with retracted fibers D = 333 sec -~ and s = 1060 S (corrected to the viscosity o~ water at 20~ With extended fibers these values change to 167 sec -I and 850 S respectively. At 20~ and intermediate concentrations of Trp insufficient to produce a saturation of the fiberextension phenomenon, a bimodal decay of birefringence was found that could only be described consistently by assuming a mixed population of particles with fibers either extended or retracted. Such a bidispers population was also found at high concentrations of Trp if the temperature was lowered to 10~ Furthermore it was found that the assumption that the presence of Trp is a necessary requirement for fiberextension is incorrect. Part of the particles extend their fibers at higher temperature (40-50 ~ C) even in the absence of Trp. This was confirmed by the finding that at this higher temperature a biological activity is found in the absence of Trp. A quantitative model is presented based on the assumption that at each Trp concentration there exists an equilibrium between the two conformations (fibers-in and fibers,out). This equilibrium is governed by the affinity of each of six accepter sites for Trp which is different for the fibers-in and fibers-out conformation. Contrary to earlier models (i) this model predicts a certain probability of fiberextension in the absence of Trp.