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PubMed1995

Tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase. Enzymatic and urea stability properties.

Early studies suggested that the Escherichia coli tryptophan synthase alpha-subunit unfolded in a two-step process in which there was a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). More recent evidence has indicated that such a structure for the intermediate seems unlikely. In this report, single Trp residues (absent in the wild-type alpha-subunit) are substituted separately for Phe residues at positions 139 (in alpha-1) and 258 (in alpha-2) to produce the F139W, F258W, and F139W/F258W mutant alpha-subunits. The UV absorbance and fluorescence properties of the F139W/F258W double mutant are identical with those of equimolar mixtures of the single mutants, suggesting that the Trp residue at each position can independently report the behavior of its respective folding unit. Each mutant alpha-subunit is wild-type enzymatically, and when UV absorbance is monitored, the urea-induced unfolding of the three tryptophan-containing alpha-subunits is virtually identical to the wild-type protein. These wild-type properties make these proteins attractive candidates for a fluorescence examination of the behavior of the individual folding units and the structure of potential intermediate(s) and as host proteins for the insertion of our existing destabilizing and/or stabilizing mutational alterations.

Base Sequence↗
PubMed1995

Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase.

The urea-induced unfolding of the Escherichia coli tryptophan synthase alpha-subunit is examined via fluorescence measurements with tryptophan-containing alpha-subunit mutants, constructed by in vitro mutagenesis. Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). Recently, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediates seems unlikely. Previously, we described the introduction of Trp residues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically and in their stability to urea. The unfolding behavior of these alpha-subunits, monitored by fluorescence intensity changes at the discrete emission lambda max for each, in both equilibrium and kinetic experiments, suggest that: (a) both folding units commence unfolding simultaneously (near 2 M urea); (b) the larger alpha-1 unit unfolds in a multistep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller alpha-2 unit unfolds in a single step event. These results are also clearly incompatible with the early proposals on the structure of the intermediate. It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of alpha-1 attached to either a completely folded or completely unfolded form of alpha-2. These results are consistent with and provide an added dimension to the recent description of the proposed structure of the intermediate.

Calorimetry↗
PubMed1997

Reversible binding interactions between the tryptophan enantiomers and albumins of different animal species as determined by novel high performance liquid chromatographic methods: an attempt to localize the D- and L-tryptophan binding sites on the human serum albumin polypeptide chain by using protein fragments.

The stereoselectivity of the reversible binding interactions between the D- and L-tryptophan enantiomers and serum albumins of different animal species and fragments of human serum albumin (HSA) was investigated by applying three novel high performance liquid chromatographic (HPLC) arrangements. The separations were performed by means of 1) an achiral (diol-bond), 2) a chiral (bovine serum albumin-bond) silica gel sorbent, and 3) a column switching technique which uses both the diol- and HSA-bond HPLC stationary phases. A polarimetric detector and/or an ultraviolet (UV) spectrophotometer were used to monitor the separation process. HPLC arrangement 3 allowed the evaluation of enantioselective binding for D- and L-tryptophan to different albumins and albumin fragments. At present, column switching can be considered the technique of the broadest applicability for investigating the reversible binding interactions between a protein and drug enantiomers.

Animals↗
PubMed1994

1,1'-Ethylidenebis(tryptophan) (Peak E) induces functional activation of human eosinophils and interleukin 5 production from T lymphocytes: association of eosinophilia-myalgia syndrome with a L-tryptophan contaminant.

This study was designed to clarify the important association between eosinophilia-myalgia syndrome (EMS) and the L-tryptophan contaminant, "Peak E." To determine the functional activation of eosinophils induced by Peak E, eosinophil cationic protein (ECP) release was examined. Peak E augumented the release of ECP from peripheral blood normodense eosinophils by degranulation. Proliferative analysis using the human eosinophilic leukemia cell line EoL-3 showed prominent cellular replication in the presence of Peak E. Moreover, Peak E upregulated interleukin 5 (IL-5) receptor levels on normodense eosinophils. Of particular interest, Peak E-stimulated human splenic T cells produced bioactive and immunoreactive IL-5. Marked induction of IL-5 mRNA in Peak E-stimulated T cells was also shown by reverse-transcriptase polymerase chain reaction (RT-PCR). In contrast, L-tryptophan without the contaminant showed none of these effects. Thus, these data suggest that Peak E might be involved in the pathogenesis of EMS through bimodal mechanism including IL-5 generation by T cells and potentiation of eosinophil functional activation.

B-Lymphocytes↗
PubMed1996

Fluorescence behavior of tryptophan residues of bovine and human serum albumins in ionic surfactant solutions: a comparative study of the two and one tryptophan(s) of bovine and human albumins.

The fluorescence behavior of two tryptophans (Trp-134, Trp-213) in bovine serum albumin (BSA) and a single tryptophan (Trp-214) in human serum albumin (HSA) was examined. The maximum emission wavelength (lambda max) was 340.0 nm for both proteins. In a solution of sodium dodecyl sulfate (SDS), the lambda max of BSA abruptly shifted to 332 nm at 1 mM SDS and then reversed to 334 nm at 3 mM SDS. The lambda max of HSA gradually shifted to 330 nm below 3 mM SDS, although it returned to 338 nm at 10 mM SDS. In contrast to this, in a solution of dodecltrimethylammonium bromide, the lambda max positions of BSA and HSA gradually shifted to 334.0 and 331.5 nm, respectively. Differences in the fluorescence behavior of the proteins are attributed to the fact that Trp-134 exists only in BSA, with the assumption that Trp-213 of BSA behaves the same as Trp-214 of HSA. The Trp-134 behavior appears to relate to the disruption of the helical structure in the SDS solution.

Animals↗
PubMed2004

Effects of a novel method of acute tryptophan depletion on plasma tryptophan and cognitive performance in healthy volunteers.

RATIONALE: Disorders associated with low levels of serotonin (5-HT) are characterized by mood and cognitive disturbances. Acute tryptophan depletion (ATD) is an established method for lowering 5-HT levels and an important tool to study the effects of reduced 5-HT on mood and cognition in human subjects. The traditional ATD method, i.e., administration of separate amino acids (AAs), has several disadvantages. The AA mixture is costly, unpalatable and associated with gastrointestinal discomfort. OBJECTIVES: The University of Maastricht developed a new and inexpensive method for ATD: a natural collagen protein (CP) mixture with low tryptophan (TRP) content. The reductions in plasma TRP after taking this CP mixture were compared with the reductions achieved taking the traditional AA mixture, and effects on memory and reversal learning were studied. METHODS: Fifteen healthy young volunteers participated in a double-blind, counterbalanced within-subject study. Reversal learning, verbal memory and pattern recognition were assessed at baseline and 3-4 h after taking the CP mixture. RESULTS: The new ATD method significantly reduced plasma TRP by 74% and the ratio between TRP and the other large AAs (TRP/LNAA) by 82%. The placebo mixture did not change these measures. Delayed recognition reaction time on the verbal learning task was increased following ATD. No other cognitive effects were found. CONCLUSIONS: The CP mixture was shown to be an efficient tool for lowering plasma TRP in humans. The validity of this method with regard to behavioral changes remains to be established in healthy, vulnerable and clinical populations.

Adult↗
PubMed1992

Albumin binding and brain uptake of 6-fluoro-DL-tryptophan: competition with L-tryptophan.

We investigated potential competition between L-tryptophan (TRP) and 6-fluoro-DL-tryptophan (6-F-TRP) for binding to albumin and for passage through the blood-brain barrier (BBB). In experiments based on equilibrium dialysis, albumin (600 microM) bound about 80% of TRP and 50% of 6-F-TRP with affinity constants (Ka) of 3.7 +/- 0.04 x 10(4) and 0.62 +/- 0.01 x 10(4) M-1, respectively. Competitive inhibition was assessed as the decrease in the apparent Ka (K' a) of TRP in the presence of 6-F-TRP, with no modification of the N value. Competition between TRP, 6-F-TRP and L-valine (VAL) for passage across the BBB was demonstrated using two approaches. When administered concomitantly with TRP or 6-F-TRP to rats, VAL decreased brain uptake of TRP and 6-F-TRP and reversed their action on serotonin. In Oldendorf's model, 6-F-TRP and VAL decreased the brain uptake of TRP.

Animals↗
PubMed2006

Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/D-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence.

Recombinant purified human sodium/D-glucose cotransporter1 (hSGLT1) was reconstituted in a functional form into phospholipid vesicles and its conformational states in the absence and presence of ligands and inhibitors were probed by intrinsic tryptophan fluorescence. In the presence of sodium, sugars increase intrinsic fluorescence (maximum 17%) in a saturable manner in the following order alpha-MDG >D-Glu approximately D-Gal >> D-Man >D-All, with no effect of L-Glu. Apparent affinities ranging from 0.65 to 10.4 mM were observed. In addition, D-Glu increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary, the transport inhibitor phlorizin decreased Trps fluorescence in a sodium-dependent manner by 50% with a red shift of 4-6 nm and decreased quencher accessibility, these effects were saturable with a high affinity of 5 microM. Furthermore, the positioning of the tryptophans in the reconstituted transporter was investigated. hSGLT1 Trps fluorescence was reduced by N-bromosuccinimide treatment maximally 25% in membranes and 65% in solution. The fluorescence was also significantly but differently quenched by the lipid-soluble spin labeled probes 5-Doxyl-phosphatidylcholine (40%) and 12-Doxyl-phosphatidylcholine (26%). Depth-calculation using the parallax method suggested a location of Trps at an average depth of 10 angstrom from the center of the bilayer. These studies demonstrate the existence of different conformational states of the membrane-embedded transporter in its glucose-free form, as sodium-glucose-carrier complex and as sodium-phlorizin-carrier complex. They further indicate that most of the Trp residues in hSGLT1 are located in hydrophobic regions of the protein or in contact with the lipid bilayer of the membrane. There, they are located close to the membrane-water interface contributing to the vectorial nature of the transporter.

Cyclic N-Oxides↗
PubMed1995

Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein.

The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins.

Base Sequence↗
PubMed1975

Changes in serum tryptophan and albumin binding of tryptophan in chlorpromazine-treated chronic schizophrenics on withdrawal and restoration of drug therapy.

Withdrawal of drug from a group of chlorpromazine-treated chronic male schizophrenics led to a significant rise in the initially low serum tryptophan concentration, and a rise in the fraction freely diffusible. These changes, which would be expected to lead to an increase in tryptophan influx into the brain, and hence to an increase in cerebral serotonin synthesis, could be related to the clinical effects of drug withdrawal.

Adult↗
PubMed1998

Activation of the Ah receptor by tryptophan and tryptophan metabolites.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a variety of hydrophobic natural and synthetic chemicals, including the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). A variety of indole-containing chemicals, such as indole-3-carbinol, indolo[3, 2-b]carbazole, and UV photoproducts of tryptophan (TRP), have previously been identified as ligands for AhR. Here we have examined the ability of endogenous metabolites of tryptophan (TRP) to bind to and activate AhR in vitro and in cells in culture. Although hydroxylated TRP metabolites were inactive, two metabolites, namely tryptamine (TA) and indole acetic acid (IAA), were shown to be AhR agonists. Not only do TA and IAA bind competitively to AhR, but they also can stimulate AhR transformation and DNA binding and induce expression of an AhR-dependent reporter gene in cells. In addition to being an AhR ligand, TA is also a competitive substrate for cytochrome P4501A1, a well-characterized AhR- and TCDD-inducible gene product. Although these compounds are relatively weak ligands, compared to TCDD, they represent some of the first endogenous hydrophilic AhR agonists identified to date.

5-Methoxytryptamine↗
PubMed2006

Covalent and noncovalent interactions for [metal(dien)nucleobase](2+) complexes with L-tryptophan derivatives: formation of palladium-tryptophan species by nucleobase substitution under biologically relevant conditions.

The interaction of the complexes [Pd(dien)(1-MeCyt)]2+ (2) and [Pd(dien)(9-EtGH)]2+ (3) with the amino acids L-tryptophan (Trp) and N-acetyltryptophan (N-AcTrp) was studied and compared with the previously studied platinum analogues [Pt(dien)(1-MeCyt)]2+ (4) and [Pt(dien)(9-EtGH)]2+ (5). Solid-state structures for 2 and 4 are reported. For the palladium complexes, the interaction is pH sensitive. Below pH 5, the noncovalent interaction with stacking between the aromatic amino acid residue and the metalated nucleobase was observed. Fluorescence quenching experiments indicated similar association constants for platinum and palladium derivatives 2-5. Unusual substitution of the model nucleobases 1-methylcytosine (1-MeCyt) and 9-ethylguanine (9-EtGH) by tryptophan was observed in the range of pH 5-11. The resulting species [Pd(dien)(Trp)]+ (6) and [Pd(dien)(N-AcTrp)]+ (7) were characterized using 1H NMR, 13C NMR, and ESI-MS spectroscopy with coordination indicated through the amino and deprotonated amido nitrogens, respectively. Complexes 6 and 7 were also obtained from a solution of [Pd(dien)Cl]+ (1) incubated with either Trp or N-AcTrp, respectively.

Crystallography, X-Ray↗
PubMed2006

5-Hydroxy-L-[beta-11C]tryptophan versus alpha-[11C]methyl-L-tryptophan for positron emission tomography imaging of serotonin synthesis capacity in the rhesus monkey brain.

The purpose of this study was to compare two positron emission tomography (PET) tracers that were developed to follow serotonin (5HT) synthesis by performing sequential PET scanning of the same rhesus monkey (n=4) on the same day. alpha-[11C]Methyl-L-tryptophan ([11C]AMT) and 5-Hydroxy-L-[beta-11C]tryptophan ([11C]HTP) are substrates in the first and second enzymatic steps, respectively, in the biosynthesis of 5HT. Regional net accumulation rate constants were derived from kinetic (two-tissue compartment model with irreversible tracer trapping) and graphic (Patlak) analyses, using the arterial plasma concentrations as input. The kinetic data analysis showed that the rate constant for the transfer of [11C]HTP into the brain (K1) was higher than that for [11C]AMT in the striatum and thalamus but was similar in other brain regions. The rate constant for tracer trapping (k3) was also higher for [11C]HTP than for [11C]AMT in the striatum (0.046+/-0.024 versus 0.019+/-0.006 min(-1)) and thalamus (0.039+/-0.013 versus 0.016+/-0.007 min(-1)). In agreement with previously reported regional HTP accumulation rates, the net accumulation rate constant (K(acc)) for [11C]HTP was also higher in these regions than in other brain regions; this is in contrast to the uniform distribution of [11C]AMT K(acc) values. This suggests that the regional net accumulation rates obtained with these two PET tracers will be of different magnitude, which might be related to the activity of each targeted enzyme.

5-Hydroxytryptophan↗
PubMed1980

Tryptophan pyrrolase in haem regulation. The relationship between the depletion of rat liver tryptophan pyrrolase haem and the enhancement of 5-aminolaevulinate synthase activity by 2-allyl-2-isopropylacetamide.

Rat liver tryptophan pyrrolase haem is maximally depleted at 30 min after administration of a 400 mg/kg dose of 2-allyl-2-isopropylacetamide. This depletion lasts for 24 h, by which time 5-aminoleevulinate synthase activity becomes maximally enhanced. 2. though the above maximum depletion of pyrrolase haem (at 0.5h) is also produced by a 100 mg/kg dose of the porphyrogen, this does not enhance synthase activity at 24 h. It and smaller doses, however, cause a smaller but earlier enhancement of synthase activity (maximum at 2 h) and produce a similarly short-lived deplation of pyrrolase haem. 3. The depletion of pyrrolase haem and the enhancement of synthase activity by the porphyrogen are inhibited by compound SKF 525-A and phenazine methosulphate, and are potentiated by nicotinamide but not by phenobarbitone. Phenazine methosulphate and nicotinamide also exert opposite effects on hexobarbital sleeping-time. 4. 2-Allyl-2-isopropylacetamde also the depletes pyrrolase haem in vitro. It does so in liver homogenates of control rats in the presence, and in those of phenobarbitone-treated rats in the absence of added NADPH. 5. A discussion of the present results in relation to previous work with other haemoproteins suggests that, whereas cytochrome P-450 (haem) is primarily involved in the production of the active (porphyrogenic) metabolite(s) of 2-allyl-2-isopropylacetamide, the haem pool used by tryptophan pyrrolase may play an important role in the effects of this compound on haem biosynthesis.

5-Aminolevulinate Synthetase↗
PubMed2004

The dynamics of conformational isomerization in flexible biomolecules. I. Hole-filling spectroscopy of N-acetyl tryptophan methyl amide and N-acetyl tryptophan amide.

The conformational isomerization dynamics of N-acetyl tryptophan methyl amide (NATMA) and N-acetyl tryptophan amide (NATA) have been studied using the methods of IR-UV hole-filling spectroscopy (HFS) and IR-induced population transfer spectroscopy (IR-PTS), which were developed for this purpose. Single conformations of these molecules were selectively excited in well-defined NH stretch fundamentals. This excess energy was used to drive conformational isomerization. By carrying out the infrared excitation early in a supersonic expansion, the excited molecules were recooled into their zero-point levels, partially refilling the hole created in the ground state population of one of the conformers, and creating gains in population in other conformers. These changes in population were detected using laser-induced fluorescence downstream in the expansion. In HFS, the IR wavelength is fixed and the UV laser tuned in order to determine where the population went following selective infrared excitation. In IR-PTS, the UV is fixed to monitor the population of a given conformation, and the IR is tuned to record the IR-induced changes in the population of the monitored conformer. Besides demonstrating the capability of the experiment to change the downstream conformational population distribution, the IR-PTS scans were used to extract two quantitative results: (i) The fractional populations of the conformers in the absence of the infrared, and (ii) the isomerization quantum yields for each of the six unique amide NH stretch fundamentals (three conformers each with two amide groups). The method for obtaining quantum yields is described in detail. In both NATMA and NATA, the quantum yields show modest conformational specificity, but only a hint of vibrational mode specificity. The prospects for the hole-filling technique for providing insight into energy flow in large molecules are discussed, leaving a more detailed theoretical modeling to the adjoining paper [Evans et al. J. Chem. Phys. 120, 148 (2004)].

Chemical Phenomena↗
PubMed1980

Influenza virus haemagglutinin: estimation of tryptophan and tyrosine content and localization of tryptophan residues.

The tryptophan and tyrosine content of the bromelain-released subtype H3 haemagglutinin (H3 BHA) of influenza virus were measured by u.v. absorption and fluorescence techniques. The values obtained (8 and 18 residuces, respectively) are in close agreement with those derived from amino acid analysis. Essentially all of th tryptophan residues are demonstrated to be localized on the surface of the BHA molecule.

Bromelains↗
PubMed1991

Quinoproteins: enzymes containing the quinonoid cofactor pyrroloquinoline quinone, topaquinone or tryptophan-tryptophan quinone.

The presently best known and largest group of quinoproteins consists of enzymes using the cofactor 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline- 4,5-dione (PQQ), a compound having a pyrrole ring fused to a quinoline ring with an o-quinone group in it. Representatives of this group are found among the bacterial, NAD(P)-independent, periplasmic dehydrogenases. Despite their high midpoint redox potential, the overall behaviour of quinoprotein dehydrogenases is similar to that of their counterparts, those using a flavin cofactor or a nicotinamide coenzyme. Apart from an exceptional Gram-positive one, the sole organisms where the presence of PQQ has really been established are Gram-negative bacteria. Evidence for the occurrence of covalently bound PQQ is lacking since it has now been shown that several enzymes previously considered to contain this prosthetic group do not in fact do so. Another group of quinoproteins, consisting of amine oxidoreductases, has a protein chain containing one of the following quinonoid aromatic amino acids: 6-hydroxy-phenylalanine-3,4-dione (TPQ) or 4-(2'-tryptophyl)-tryptophan-6,7-dione (TTQ). There is no doubt that these o-quinones play a role as cofactor, in the case of TPQ in prokaryotic as well as eukaryotic amine oxidases. It appears, therefore, that a novel class of amino-acid-derived cofactors is emerging, ranging from the free radical form of tyrosine and tryptophan to those containing a dicarbonyl group (like the already known pyryvoyl group and the o-quinones here described.

Coenzymes↗