Presence Of Tryptophan Research Articles

Identification of tyrosine in the presence of tryptophan using Cd 2+-enriched colloidal CdS nanoparticles: A fluorescence spectroscopic study

A simpler identification method of tyrosine in the presence of tryptophan using CdS nanoparticles by conventional spectroscopic technique is proposed. Effect of both sulfide-enriched CdS as well as Cd 2+-enriched CdS on tryptophan is investigated through absorption and emission spectroscopy. Quenching of tryptophan emission obeyed Stern–Volmer relation and was found to be independent of temperature, indicating a possible static quenching. The time-resolved fluorescence decay of tryptophan was minimally affected by sulfide-enriched CdS as well as Cd 2+-enriched CdS nanoparticles, suggesting quenching to be static. In the presence of Cd 2+-enriched CdS nanoparticles, the emission of tryptophan in phosphate buffer shows a typical spectral broadening along with a long wavelength increase in fluorescence emission. Additionally, spectra followed a typical isoemissive point at 440 nm when tryptophan alone was there. Similarly, isoemissive point at 340 nm was observed in the case of tyrosine. However, a further red shift of isoemissive point (470 nm) in the mixture of both tyrosine and tryptophan was observed. This observation might make Cd 2+-enriched CdS nanoparticles useful for using as marker for tyrosine in the presence of tyrptophan.

Indoleamine 2,3 dioxygenase and human leucocyte antigen‐G inhibit the T‐cell alloproliferative response through two independent pathways

Both human leucocyte antigen (HLA)-G and indoleamine 2,3 dioxygenase (IDO) are key molecules involved in immune tolerance. HLA-G is a non-classical HLA class I molecule that can be expressed in both membrane-bound (HLA-G1) and soluble (HLA-G5) forms, both of which exhibit tolerogenic properties via interaction with inhibitory receptors present on natural killer (NK) cells, T cells and antigen-presenting cells (APC). IDO is an enzyme that acts by depleting the surrounding microenvironment of the essential amino acid, tryptophan, thereby inhibiting T-cell proliferation. Our present study was aimed at analysing the potential link that may exist between IDO and HLA-G. Our results showed that during allogeneic reactions, soluble HLA-G expression was not regulated by the addition of IDO substrate (i.e. tryptophan), metabolite (i.e. kynurenine) or inhibitor (i.e. 1-methyl-tryptophan), that IDO activity was not altered by HLA-G5 treatment, and that HLA-G5-mediated inhibition of the T-cell alloproliferative response was neither affected by the presence of tryptophan and kynurenine nor reversed after IDO activity blockage, demonstrating that HLA-G5 can exert its function in the absence of functional IDO. Similarly, inhibition of the T-cell alloresponse, induced by HLA-G1-expressing antigen-presenting cells, was not altered by IDO metabolites or inhibitor. Taken together, these findings show that the function and expression of IDO and HLA-G5 are not mutually influenced, but rather inhibit the T-cell alloproliferative response through two independent pathways. IDO and HLA-G are thus complementary for inducing and maintaining immune tolerance in physiological (pregnancy) and pathological (tumour and allograft) situations.

Regulation of tryptophan operon expression in the archaeon Methanothermobacter thermautotrophicus.

Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNA(Trp) available to translate the second codon of the trpY mRNA.

Electron Transfer Chemistry between DNA and DNA-Binding Tripeptides

A DNA system consisting of pyrene-modified oligonucleotides and nitrobenzoate (Nb)-modified DNA-binding tripeptides has been applied to study electron-transfer processes through the DNA-peptide interface. 5-(Pyren-1-yl)-2'-deoxyuridine (Py-dU) has been used as the photoinducible charge generator. Upon excitation at 350 nm, a pyrene-like excited state (Py-dU) is formed which undergoes an electron transfer yielding the charge-separated state which is the contact ion pair Py(*)(+)-dU(*)(-). The subsequent electron shift from dU(*)(-) into the base stack competes with charge recombination and can be probed chemically by trapping the electron at the 5-bromo-2'-deoxyuridine (Br-dU) group leading to strand cleavage which can be quantified by HPLC analysis. Several Nb-modified DNA-binding tripeptides influence these DNA-mediated electron-transfer processes as shown by fluorescence spectroscopy experiments. Fluorescence quenching can occur primarily through a reductive electron-transfer process in which the Nb group traps the electron thermodynamically from the contact ion pair Py(*)(+)-dU(*)(-). Moreover, our results indicate that, once the negative charge has been trapped on the peptide, oxidative processes from Py(*)(+) take place resulting in an enhanced and nonspecific strand degradation of the Py-dU-modified duplexes. The latter type of strand cleavage can be inhibited by the presence of tryptophane or tyrosine as part of the peptides. Most remarkably, DNA-binding tripeptides, which bear both the Nb and the tryptophan/tyrosine moiety, are able to trap both the negative and the positive charge from the contact ion pair Py(*)(+)-dU(*)(-).

Bacillus Pumilus TX703 유래 Xylanase의 활성에 관여하는 아미노산 잔기의 확인

The purified xylanase from Bacillus pumilus TX703 was modified with various chemical modifiers to determine the active sites of the enzyme. Treatment of the enzyme with group-specific reagents such as carbodiimide or N-bromosuccinimide resulted in complete loss of enzyme activity. These results assumed that these reagents reacted with glutamic acid or aspartic acid and tryptophan residues located at or near the active site. In each case, inactivation was performed by pseudo first-order kinetics. Inhibition of enzyme activity by carbodiimide and W-bromosuccinimide showed non-competitive and competitive inhibition type, respectively. Addition of xylan to the enzyme solution containing N-bromosuccinimide prevented the inactivation, indicating the presence of tryptophan at the substrate binding site. Analysis of kinetics for inactivation showed that the loss of enzyme activity was due to modification of two glutamic acid or aspartic acid residues and single tryptophan residue.

Mechanisms of secondary structure breakers in soluble proteins.

Breaking signals of secondary structure put strong limitations on the tertiary structures of proteins. In addition to proline and glycine clusters, which are well-known secondary structure breakers, clusters of amphiphilic residues were found to be a novel type of secondary structure breaker. These secondary structure breakers were found to depend on specific environmental factors. Such conditions included the average hydrophobicity, the helical periodicity, the density of serine and threonine residues, and the presence of tryptophan and tyrosine clusters. Principal component analysis of environmental factors was conducted in order to identify candidate breakers in the secondary structure breaking regions. Predicted breakers were located in breaking regions with an accuracy of 72%. Taking the loop core into consideration, almost 90% of the predicted breakers were located in the loop segments. When the migration effect of the breaking point was taken into account, the loop segments with the predicted breakers covered two thirds of all loop segments. Herein, the possibility of secondary structure prediction based on secondary structure breakers is discussed. The system of the present method is available at the URL: http://bp.nuap.nagoya-u.ac.jp/sosui/sosuibreaker/sosuibreaker_submit.html.

Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea

Free-living nitrogen fixing bacteria were isolated from rhizosphere of seven different plant namely sesame, maize, wheat, soybean, lettuce, pepper and rice grown in Chungbuk Province, Korea. Five isolates with nitrogenase activity above 150nmol(-1) mg(-1) protein were identified based on, phenotypic and 16S rDNA sequences analysis. The strains were identified as Stenotrophomonas maltophilia (PM-1, PM-26), Bacillus fusiformis (PM-5, PM-24) and Pseudomonas fluorescens (PM-13), respectively. All the isolates produced indole-3-acetic acid (IAA), in the presence of tryptophan, ranging from 100.4 microg ml(-1) (PM-13) to 255 microg ml(-1) (PM-24). The isolate PM-24 (Bacillus fusiformis) exhibiting highest nitrogenase activity (3677.81 nmol h(-1) mg(-1) protein) and IAA production (255microg ml(-1)) has a promising potential for developing as a plant growth promoting rhizobacteria.

Characterization of serotonin transporter in blood lymphocytes of rats. Modulation by in vivo administration of mitogens

Serotonin transporter sites were characterized in blood lymphocytes of rats. Pharmacological characteristics of drug interactions were in concordance with recent studies in nervous and human immune cells. The potency order of inhibition of [ 3H]paroxetine binding was imipramine>citalopram>alaproclate>serotonin. Selective inhibitors of dopamine or noradrenaline transporters did not inhibit it. The specific binding of [ 3H]paroxetine was higher at intermediate than at low concentrations, and the plot of free vs. specific binding had a sigmoid shape. The affinity constant or K d, 1.77 nM, was in close agreement with data obtained from kinetic studies ( K d=1.33 nM), which evidences that the equilibrium was reached. In addition, serotonin transporter was evaluated by lipopolysaccharide or concanavalin A administration in vivo (0.1 mg/kg, i.p., 18 h). After the treatment with lipopolysaccharide, no changes were observed in the numbers of sites or B max or in the affinity, K d. The treatment with concanavalin A showed a significant reduction in B max and reduction in K d. Additionally, serotonin and 5-hydroxyindoleacetic acid levels were determined in plasma and lymphocytes by high-performance liquid chromatography. Treatment with lipopolysaccharide produced a significant increased of serotonin levels in lymphocytes without changes in 5-hydroxyindoleacetic acid level; in plasma, it produced an increase in serotonin and 5-hydroxyindolacetic acid levels. In addition, serotonin synthesis was evaluated by adding 300 μM of tryptophan in the medium, which significantly increased serotonin levels in control lymphocytes. Moreover, the concentrations of 5-hydroxyindoleacetic acid was enhanced significantly, both in plasma and lymphocytes in the presence of tryptophan after treatment with lipopolysaccharide. The administration of concanavalin A significantly decreased plasma levels of serotonin, as well as the concentrations of serotonin and 5-hydroxyindoleacetic acid in lymphocytes. These results demonstrate the presence of serotonin transporter in lymphocytes of rat blood, the capacity for serotonin synthesis in lymphocytes, and the modulation of these parameters by systemic administration of mitogens. The findings of this work contribute to understanding the immunological role of serotonin and the communication of immune and nervous systems.

Biosynthesis of Indole-3-Acetic Acid by the Gall-inducing Fungus Ustilago esculenta

Ustilago esculenta incites the formation of an edible gall and prevents inflorescence and seed production in the aquatic perennial grass, Zizania latifolia. As compared to the healthy tissues, the edible galls had higher amounts of indole-3-acetic acid (IAA), which could be synthesized from the host plant and/or the fungal pathogen. In this study we investigated the ability for IAA production by U. esculenta. The production of IAA in c ulture by U. esculenta was solely dependent on the presence of tryptophan. The addition of thiamine (vitamin B) to medium greatly enhanced fungal growth, whereas IAA production was completely 1 inhibited. Maximum amount of IAA (ca.1.0 µg mL ) was obtained after 8-day incubation. The production of 1 IAA was highly correlated with the amount of tryptophan. The optimum temperature for IAA production ranged from 20 to 25oC. However, IAA production was significantly reduced when fungus was grown in the fluctuated temperatures, indicating that a constant temperature has a profound effect on IAA production. The corn smut pathogen U. maydis also synthesized IAA using tryptophan and accumulated a maximum amount of IA A (ca.1.2 µg mL ) at day 3, then production was declined steadily thereafter. In contrast, the sugarcane smut 1 pathogen U. scitaminea produced less amount of IAA (ca.0.53 µg mL ) compared to other two species. In 1 addition to tryptophan, U. esculenta apparently could convert indole-3-acetamide (IAAld), indole-pyruate (IPA) and indole-lactic acid (ILA) into IAA. However, indole-3-acetamide (IAM) was evidently not a suitabl e precursor for IAA production. The results suggest that biosynthesis of IAA in U. esculenta from tryptophan proceeds through IPA and IAAld.

Efeito da inoculação de Azospirillum e Herbaspirillum na produção de compostos indólicos em plântulas de milho e arroz

Bactérias fixadoras de nitrogênio podem contribuir para o crescimento vegetal pela produção de auxinas. Os objetivos deste trabalho foram quantificar a produção de hormônios de crescimento por estirpes de bactérias fixadoras de nitrogênio e avaliar o efeito da inoculação destas estirpes em plântulas de milho e trigo. Todas as estirpes avaliadas começaram a produzir indóis no final da fase logarítmica. Houve efeito da adição de diferentes concentrações de triptofano ao meio de cultivo no aumento da produção de indóis até o nível de 200 µM. Nas estirpes de Azospirillum, as formas de N: NH4Cl, (NH4)2SO4, NH4H2PO4 e NH4NO3 estimularam a produção de indóis em níveis baixos. A adição de KNO3, NaNO3 e KNO2 inibiu a produção de indóis em todas as bactérias testadas e no crescimento de células de Azospirillum. Os efeitos da inoculação foram também comparados com concentrações crescentes do ácido 3-indolacético, KNO3 e triptofano. Em condições axênicas, a elevada produção de indóis reduz o comprimento das raízes e colmos na presença de triptofano, especialmente quando submetidas à inoculação de Azospirillum.

Occurrence of Gluconacetobacter diazotrophicus in tropical and subtropical plants of Western Ghats, India

Endophytic bacteria were isolated from the tissues of surface sterilized roots, stems, and leaves of fifty different crop plants. Phenotypic, biochemical tests and species-specific PCR assay permitted identification of four isolates of Gluconacetobacter diazotrophicus from root tissues of carrot ( Daucus carota L.), raddish ( Raphanus sativus L.), beetroot ( Beta vulgaris L.) and coffee ( Coffea arabica L.). Further the plant growth promoting traits such as nitrogenase activity, production of phytohormone indole acetic acid (IAA), phosphorus and zinc solubilization were assessed. Significant nitrogenase activity was recorded among the isolates and all the isolates produced IAA in the presence of tryptophan. Though all the four isolates efficiently solubilized phosphorus, the zinc solubilizing ability differed among the isolates.

Interaction of the trp RNA-binding Attenuation Protein (TRAP) with Anti-TRAP

The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis. In the presence of tryptophan, TRAP is activated to bind to the 5′-leader region of the trp mRNA resulting in termination prior to the structural genes. In addition, accumulation of uncharged tRNA Trp induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function. Both of these proteins consist of oligomers of identical subunits. Here, we characterize the self-association of each of these proteins and the TRAP–AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation. TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan. Tryptophan binding induces a conformational change in TRAP. AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of ∼3×10 14 M −3. About 20% of the trimer is incompetent to form dodecamer. The AT equilibrium is slow on the time-scale of the velocity experiment. Formation of TRAP–AT complexes occurs only in the presence of tryptophan. A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity. At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected. A model for the structure of the complex is proposed.

Adduct formation by photo-induced electron transfer between photo-oxidising Ru(ii) complexes and tryptophan

The (3)MLCT excited state of Ru(II) complexes with 1,4,5,8-tetraazaphenanthrene (TAP) ligands in the presence of tryptophan gives rise to an electron transfer process from the amino acid with subsequent formation of an adduct between the two partners, strongly influenced by oxygen.

Fluorogenic peptide sequences--transformation of short peptides into fluorophores under ambient photooxidative conditions.

Long-lived proteins are susceptible to nonenzymatic chemical reactions and the evolution of fluorescence; however, little is known about the sequence-dependence of fluorogenesis. We synthesized a library of over half a million octapeptides and exposed it to light and air in pH 7.4 buffer to identify fluorogenic peptides that evolve under mild oxidative conditions. The bead-based peptide library was composed of the general sequence H(2)N-Ala-(Xxx)(6)-Ala-resin, where Xxx was one of nine representative amino acids: Asp, Gly, His, Leu, Lys, Pro, Ser, Trp, and Tyr. Next, we selected five highly fluorescent beads from the library and subjected them to microsequencing, revealing the sequence of the unreacted peptide. All five of the fluorogenic sequences were ionic; lacked Tyr, His, and Leu; and most of the sequences contained only one Trp. We then synthesized the five soluble peptides corresponding to the fluorogenic peptide sequences and exposed them to photooxidative conditions. In general, the soluble peptides reacted slowly, generating nonfluorescent monooxygenated and dioxygenated products. However, one peptide (H(2)N-AlaLysProTrpGlyGlyAspAla-CONH(2)) evolved into a highly fluorescent photoproduct as well as a nonfluorescent monooxygenated photoproduct. The fluorescent photoproduct consisted of a 2-carboxy-quinolin-4-yl moiety fused to the N-terminus of GlyGlyAspAla. The formation of this photoproduct requires cleavage of the peptide backbone and a dramatic reorganization of tryptophan. This work demonstrates that sequencing unreacted peptide on beads can reveal sequences with unique nonenzymatic reactivity. The study also confirms that peptide fluorogenesis is dependent on sequence and not merely on the presence of tryptophan. The potential importance of fluorogenic peptide sequences is two-fold. First, fluorogenic sequences that arise through mutation could prove to be hot spots for human aging. Second, fluorogenic sequences, particularly those compatible with intracellular conditions, may serve as fluorescent tags for proteins or as fluorescent biomaterials.

Indole derivatives produced by the fungus Colletotrichum acutatum causing lime anthracnose and postbloom fruit drop of citrus

Postbloom fruit drop (PFD) of citrus and Key lime anthracnose (KLA) are caused by Colletotrichum acutatum. Both fungal isolates can infect flower petals, induce young fruit abscission and result in severe yield loss on many citrus cultivars. Previous studies revealed that infection of citrus flowers by C. acutatum caused higher levels of indole-3-acetic acid (IAA), which could be synthesized from the host plant and/or the fungal pathogen. The ability for IAA production by C. acutatum isolates was investigated. Similar to many microorganisms, the production of indole compounds in the medium by C. acutatum was dependent solely on the presence of tryptophan (Trp). In total, 14 PFD and KLA fungal isolates were tested, and revealed that they all were capable of utilizing Trp as a precursor to synthesize IAA and other indole derivatives. High-performance liquid chromatography analysis and chromogenic stains after a fluorescence thin-layer chromatography separation unambiguously identified IAA, tryptophol (TOL), indole-acetaldehyde, indole-acetamide (IAM), indole-pyruvic acid, and indole-lactic acid (ILA) from cultures supplemented with Trp. The data suggest that C. acutatum may synthesize IAA using various pathways. Interestingly, increasing Trp concentrations drastically increased the levels of TOL and ILA, but not IAA and IAM. The ability of C. acutatum to produce IAA and related indole compounds may in part contribute to the increased IAA levels in citrus flowers after infection.

Determination of the amino acid tryptophan and the biogenic amine tryptamine in foods by the heavy atom induced-room temperature phosphorescence methodology.

Very simple and selective methods are presented to determine the amino acid tryptophan (Trp) and the biogenic amine tryptamine (Tryp), both compounds with an indole-type molecular structure by the methodology named Heavy Atom Induced-Room Temperature Phosphorescence (HAI-RTP) which constitutes the first time that HAI-RTP has been used to detect compounds with non-naphthalenic structures in their molecules. Different variables affecting the phosphorescence signal (heavy atom perturber and sodium sulfite concentration) were carefully studied. The analytical curves give a linear dynamic range of 15-100 ng ml(-1) and a detection limit of 4 ng ml(-1) for Trp and 94-400 ng ml(-1) and 28 ng ml(-1) for Tryp. The methods have been successfully applied to the analysis of complex food matrices such as the presence of tryptophan in yoghurt and tryptamine in bottled beer. A single alkaline hydrolysis to release Trp from yoghurt proteins and two methods for extracting Tryp from beer samples are proposed and optimised. A total Trp content of 374 mg of Trp per kg of yoghurt was quantified by the standard addition method of calibration and a recovery of 90% was obtained for 250 ng ml(-1) of Tryp in spiked non-alcoholic beer samples.

Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces sp. KJ-72.

A bacterial strain capable of degrading medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was isolated from a soil sample. This organism, which was identified as Streptomyces sp. KJ-72, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated on MCL-PHAs. The extracellular MCL-PHA depolymerase of the organism was purified to electrophoretic homogeneity by ion exchange column chromatography and gel filtration. The enzyme consisted of a monomeric subunit having a molecular mass of 27.1 kDa and isoelectric point of 4.7. The maximum activity was observed at pH 8.7 and 50 degrees C. The enzyme was sensitive to N-bromosuccinimide and acetic anhydride, indicating the presence of tryptophan and lysine residues in the catalytic domain. The enzyme was able to hydrolyze various chain-length p-nitrophenyl esters of fatty acids and polycaprolactone as well as various types of MCL-PHAs. However, lipase activity of the enzyme was not detected. The main hydrolysis product of poly(3-hydroxyheptanoate) was identified to be the dimer of 3-hydroxyheptanoate.

Evolution of phage with chemically ambiguous proteomes

BackgroundThe widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality.ResultsThe bacteriophage Qβ initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan. After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan. Seven mutations were fixed throughout the phage in two independent lines of descent. None of the mutations changed a tryptophan residue.ConclusionsA relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins. These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon. It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates.

The Photophysical Properties of 6-Azaindole

The biosynthetic incorporation of tryptophan analogues into proteins using plasmid expression systems has been shown to offer advantages in fluorescence spectroscopic studies of the molecular details of the interacting segments of protein−protein complexes. There are two underlying concepts of this approach. The first is based on a close structural similarity between the analogue and the native tryptophan. The second is that the analogue has an extended red absorbance permitting the selective excitation in the presence of tryptophan. The reported intense absorbance band of 6-azatryptophan at 325 nm would appear to offer a unique opportunity of using this analogue in such studies. In this work, we present the photophysical characterization of the excited state behavior of 6-azatryptophan through a complete study of the cognate indole moiety, 6-azaindole. It was found that the absorbance at 320 nm for 6-azaindole and 325 nm for 6-azatryptophan was due to the protonated form in which both ring nitrogens are protonated. The protonation equilibrium for 6-azaindole had a pKa value of 8. The fluorescence of 6-azaindole was centered at 380 nm. It was found that the neutral form of 6-azaindole was nonfluorescent in nonpolar solvents. In aqueous solution at pH values between 6.5 and 10, the fluorescence maximum of 6-azaindole remained at 380 nm, and the intensity decreased as the pH increased. Above pH 10, there was increased fluorescence on the low-energy side of the spectrum until at pH 14 the fluorescence spectrum showed a single maximum at 440 nm. The observations of the collective experiments in a variety of solvent conditions and pH values, including time-resolved fluorescence measurements, permitted a consistent assignment of each of the fluorescence species together with a rationalization of the excited-state processes that result from the excitation of either the protonated form or the neutral N1−H tautomer. The 440 nm fluorescence was attributed to the fluorescence from the excited state of the N6−H tautomer, a species that formed from the protonated excited state molecule that had previously been formed from initial excitation of the N1−H ground state. The work reports determinations of the excited-state pKa values of each of the three species. The relative energy values of each of the ground-state molecules and excited-state species could be estimated. The relevance of the photophysics of 6-azaindole to protein structural studies was demonstrated by the biosynthetic incorporation of 6-azatryptophan into the Y99W mutant of rat calmodulin and the observation of 6-azaindole fluorescence from this protein.

Ribosome Regulation by Protein Products

The leader peptide regulation of the Escherichia coli tryptophanase operon tnaC has a stop codon at position 25 that yields a 24-residue peptide in the absence of inducer. However, in the presence of tryptophan, the leader peptide stays attached to the stalled ribosome as a peptidyl tRNA, and termination is blocked. By altering codons and spacing between codons in the leader peptide, Gong and Yanofsky (see the Perspective by Sachs and Geballe) show that the sequence of the nascent peptide can regulate the translating ribosome, perhaps by creating a ribosome binding site for free tryptophan. Hence, peptides are not only products of translation, but they can also control ribosome movement during translation. F. Gong, C. Yanofsky, Instruction of translating ribosome by nascent peptide. Science 297 , 1864-1867 (2002). [Abstract] [Full Text] M. S. Sachs, A. P. Geballe, Sense and sensitivity--Controlling the ribosome. Science 297 , 1820-1821 (2002). [Abstract] [Full Text]