Origin and behavior of deuteron spin echoes in selectively labeled amino acids, myoglobin microcrystals, and purple membranes

We have obtained deuterium NMR spin-echo spectra of crystalline DL-[gamma-2H6]valine, [S-methyl-2H3]-methionine, cyanoferrimyoglobin from sperm whale (Physeter catodon), containing deuteriomethyl groups at methionine-55 and methionine-131, and [gamma-2H6]valine-labeled bacteriorhodopsin in the purple membrane of Halobacterium halobium R1. By using 90-tau-beta 90 degrees (XY) and 90-tau-beta 0 degrees (XX) pulse sequences and observing the dependence of the spin-echo amplitude upon the interpulse spacing tau, we have determined that the so-called "quadrupole echoes" obtained in these typical selectively deuterated condensed-phase biological systems are in fact strongly modulated by proton-deuteron and deuteron-deuteron dipolar interactions. The two amino acids and the protein crystals behaved as typical organic solids, with no evidence of "liquid-like" behavior, even in the presence of excess water (in the case of the ferrimyoglobin crystals). However, with the valine-labeled bacteriorhodopsin, the tau-dependence of XY echoes as a function of temperature emphasized the "solid-like" behavior of the membrane "matrix", while the basic nature of the spin-echo response for the narrow central component of the spectrum clearly indicated the very "fluid" or "mobile" nature of a series of residues that are shown elsewhere [Keniry, M., Gutowsky, H. S., & Oldfield, E. (1984) Nature (London) 307, 383-386] to arise from the membrane surface. Our results thus suggest that such NMR methods may yield useful information on side-chain dynamics complementary to that of line-shape and spin-lattice relaxation time analyses.     

Nuclear magnetic resonance studies of amino acids and proteins. Side-chain mobility of methionine in the crystalline amino acid and in crystalline sperm whale (Physeter catodon) myoglobin

We have obtained deuterium (2H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T1) of L-[epsilon-2H3]methionine, L-[epsilon-2H3]methionine in a D,L lattice, and [S-methyl-2H3]methionine in the crystalline solid state, as a function of temperature, in addition to obtaining 2H T1 and line-width results as a function of temperature on [epsilon-2H3]methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering [Rothgeb, T. M., & Oldfield, E. (1981) J. Biol. Chem. 256, 1432-1446]. The results indicate that in the L-amino acid, methyl rotation having an activation energy (delta E) of 8.3 +/- 1 kJ dominates T1 at low temperatures (less than or equal to 10 degrees C), while at higher temperatures an additional large-amplitude side-chain motion occurs which causes changes in the 2H NMR line shape and T1. This motion is inhibited in the D,L lattice, indicating that lattice effects may have a strong effect on the mobility of anhydrous amino acids in the solid state. Further substitution at S delta to form the sulfonium salt [S-methyl-2H3]-methionine causes a large increase in delta E, to 15.9 +/- 2 kJ, a value comparable to the 14-16 kJ found in valine and leucine, which contain the structurally similar isopropyl moiety. These results suggest that the very low barriers to methyl rotation in the methionine side chain are due to long C-S bond lengths and the presence of only two substituents on sulfur, while the anomalous high-temperature behavior is due to a lattice-packing effect. 2H T1 results with methionine-labeled myoglobin are complex, reflecting the presence of fast large-amplitude side-chain motions, in addition to rapid methyl rotation. Our data indicate that Met-55 and Met-131 are motionally inequivalent in crystalline cyanoferrimyoglobin, in contrast to solution NMR results. We have also recorded 13C cross-polarization "magic-angle" sample-spinning NMR spectra of [epsilon-13C]methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution. Cross-polarization transfer rates and proton rotating-frame relaxation time results again indicate that Met-55 and Met-131 are motionally inequivalent in the solid state, and the TCH data indicate that Met-55 is more solidlike. However, we find that 13C chemical shifts in solution and those in the crystalline solid state are in very close agreement, suggesting that the average solution and crystal conformations are the same, in the area of Met-55 and Met-131.     

Biochemical characterisation of Nicotiana plumbaginifolia auxotrophs that require branched-chain amino acids

The biochemical lesions in six amino acid-requiring auxotrophic lines of Nicotiana plumbaginifolia have been investigated, by means of feeding experiments with [¹⁴C] and unlabelled substrates, and enzyme analysis. Three of the lines require isoleucine for growth, are unable to synthesise 2-oxobutyrate in vivo and have no detectable threonine dehydratase (E.C.4.2.1.16) in vitro. The other three lines require (isoleucine + valine), accumulate [¹⁴C] dihydroxymethylvalerate when fed [¹⁴C]-L-threonine, and contain no detectable dihydroxyacid dehydratase (E.C.4.2.1.9).     

Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts

The metabolism of ¹⁴C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [¹⁴C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-¹⁴C]diaminopimelic acid was readily converted into [¹⁴C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO₂ fixation.     

Permeability of amino acids into liposomes.

1. A simple and rapid assay for the measurement of permeability of amino acids into liposome membrane was carried out by using the liposomes trapping D-amino acid oxidase (D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) inside the membrane. 2. Permeability of amino acids into liposomes depended on the lipid composition of the membrane. Permeability of amino acids into phosphatidylcholine-cholesterol liposomes depended critically on temperature. 3. Permeability also depended on the structure of amino acids. The order of permeability was norvaline greater than isoleucine greater than leucine greater than phenylalanine greater than tryptophan greater than methionine greater than tyrosine, valine greater than threonine greater than serine greater than alanine greater than glycine.     

Use of 3H and 14C double-labeled glucose to assess in vivo pathways of amino acid biosynthesis in Escherichia coli.

3H and 14C tracing data concerning amino acid biosynthetic pathways in Escherichia coli K12 are presented. Thirteen acidic and neutral amino acids were isolated from protein hydrolysates of wild type E. coli K12 grown aerobically or anaerobically in the presence of [U-14C]glucose together with [1-3H]glucose, [3-3H]glucose, [4-3H]glucose, or [6-3H]glucose. The observed 3H/14C counts of the amino acids were compared with the ratios expected on the basis of the input substrate specific activities and present understanding of biosynthetic pathways. For nine amino acids, serine, valine, leucine, threonine, isoleucine, glycine, glutamate, proline, and phenylalanine, the agreement between anticipated and observed specific activities was satisfactory. For the remaining four, methionine, alanine, aspartate, and (in cells labeled with [3-3H]glucose) tyrosine, the anticipated and observed specific activities differed markedly. For alanine, aspartate, and tyrosine, the differences are probably due to exchange of tritium in the course of biosynthesis; for methionine, it may be that there is a principle source of the methyl group other than carbon 3 of serine.     

Nuclear magnetic resonance studies of amino acids and proteins. Rotational correlation times of proteins by deuterium nuclear magnetic resonance spectroscopy

We show that measurement of the spin-lattice (T1) and spin-spin (T2) relaxation times (or line widths) of irrotationally bound 2H nuclei in macromolecules undergoing isotropic rotational motion outside of the extreme narrowing limit (i.e., for the case omega 02 tau R2 much greater than 1) permits determination of both the rotational correlation time (tau R) of the macromolecule and the electric quadrupole coupling constant (e2qQ/h) of the 2H label. The technique has the advantage over 13C nuclear magnetic resonance (NMR) that no assumptions about bond lengths (which appear to the sixth power in 13C relaxation studies) or relaxation mechanisms need to be made, since relaxation will always be quadrupolar, even for aromatic residues at high field. Asymmetry parameter (eta) uncertainties are shown to cause negligible effects on tau R determinations, and in any case it is shown that both e2qQ/h and eta may readily be determined in separate solid-state experiments. By way of example, we report 2H NMR results on aqueous lysozyme (EC 3.2.1.17) at 5.2 and 8.5 T (corresponding to 2H-resonance frequencies of 34 and 55 MHz). Interpretation of the results in terms of the isotropic rigid-rotor model yields e2qQ/h values of approximately equal to 170 or approximately equal to 190 kHz, respectively, for the imidazolium and free-base forms of [epsilon 1-2H] His-15 lysozyme in solution, in excellent agreement with e2qQ/h values of approximately 167 and approximately 190 kHz obtained for the free amino acids in the solid state. In principle, the method may in suitable cases permit comparison between the dynamic structures of proteins in solution and in the crystalline solid state.     

Modulation of branched-chain amino acid oxidation in rat hemidiaphragms in vitro by glucose and ketone bodies

Branched-chain amino acid metabolism in hemidiaphragms from 40 h-starved rats is influenced by the provision of glucose as co-substrate. Glucose inhibits 14CO2 production from [l-14C]valine and [U-14C]valine but stimulates 14CO2 production from [l-14C]leucine, [U-14C]leucine and [U-14C]isoleucine. In the presence of glucose, ketone bodies inhibit alanine release and 14CO2 production from [l-14C]valine, [l-14C]leucine and [U-14C]isoleucine, but inhibition is not observed in the absence of glucose as cosubstrate. Glucose-dependent inhibition by ketone bodies of branched-chain amino acid oxidation via inhibition of the branched-chain 2-oxo acid dehydrogenase complex or branched-chain amino acid aminotransferase may account in part for the reported hypoalanaemic action of ketone bodies in vivo.     

Homocysteine toxicity in Escherichia coli is caused by a perturbation of branched-chain amino acid biosynthesis

In Escherichia coli the sulfur-containing amino acid homocysteine (Hcy) is the last intermediate on the methionine biosynthetic pathway. Supplementation of a glucose-based minimal medium with Hcy at concentrations greater than 0.2 mM causes the growth of E. coli Frag1 to be inhibited. Supplementation of Hcy-treated cultures with combinations of branched-chain amino acids containing isoleucine or with isoleucine alone reversed the inhibitory effects of Hcy on growth. The last intermediate of the isoleucine biosynthetic pathway, alpha-keto-beta-methylvalerate, could also alleviate the growth inhibition caused by Hcy. Analysis of amino acid pools in Hcy-treated cells revealed that alanine, valine, and glutamate levels are depleted. Isoleucine could reverse the effects of Hcy on the cytoplasmic pools of valine and alanine. Supplementation of the culture medium with alanine gave partial relief from the inhibitory effects of Hcy. Enzyme assays revealed that the first step of the isoleucine biosynthetic pathway, catalyzed by threonine deaminase, was sensitive to inhibition by Hcy. The gene encoding threonine deaminase, ilvA, was found to be transcribed at higher levels in the presence of Hcy. Overexpression of the ilvA gene from a plasmid could overcome Hcy-mediated growth inhibition. Together, these data indicate that in E. coli Hcy toxicity is caused by a perturbation of branched-chain amino acid biosynthesis that is caused, at least in part, by the inhibition of threonine deaminase.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Effect of cypermethrin on the free amino acids pool in an organophosphorus-insecticide-resistant and a susceptible strain of Tribolium castaneum

1. Proline was found to be the major component of CTC-12 (44%) and FSS II (45%) strain.2. The cypermethrin treatment resulted in an increase in most of the amino acids of sixth instar larvae and all amino acids of adult beetles of CTC 12 strain.3. In the susceptible strain (FSS II), however, the tyrosine, phenylalanine and arginine increased, whereas serine, proline, glycine, alanine, valine, isoleucine, leucine and lysine were decreased significantly in the sixth instar larvae.4. In the FSS II adult beetles, only aspartic acid increased, while other amino acids either decreased (threonine, proline, glycine, alanine, valine, methionine, isoleucine, tyrososine, lysine, arginine) or remained unaffected (serine, glutamic acid, leucine, phenylalanine, histidine).     

The role of phosphoenolpyruvate carboxykinase in muscle alanine synthesis.

3-Mercaptopicolinic acid (3-MPA) is reportedly a specific inhibitor of phosphoenolpyruvate (PEP) carboxykinase and has hitherto been used accordingly to elucidate the metabolic role of PEP carboxykinase in vitro and in vivo. We show that 3-MPA has multiple effects on intermediary metabolism in hemidiaphragms from 40 h-starved rats. It decreases the release of lactate + pyruvate and alanine in hemidiaphragms provided with no added substrate or with valine, leucine or isoleucine. Moreover, irrespective of the substrate provided (none, valine, leucine, isoleucine, glucose, acetate, oleate), 3-MPA decreases the [lactate]/[pyruvate] ratio. 3-MPA is without effect on 14CO2 production from [U-14C]valine, [1-14C]valine, [1-14C]leucine, [U-14C]isoleucine or [1-14C]oleate, but stimulates 14CO2 production from [U-14C]glucose and [1-14C]pyruvate and inhibits 14CO2 production from [1-14C]acetate. Glycolytic flux (measured as 3H2O formation from [5-3H]glucose) is stimulated by 3-MPA. It is concluded that 3-MPA has site(s) of actions other than PEP carboxykinase and that the putative role of PEP carboxykinase in alanine synthesis de novo in skeletal muscle from tricarboxylic acid-cycle intermediates and related amino acids requires reappraisal.     

Metabolism of amino acids in Ascaridia galli: transamination

Ascaridia galli, using 2-oxoglutarate as an acceptor, transaminates alanine and aspartate at significantly high rates. Among other amino acids valine, phenylalanine, leucine, isoleucine, arginine, tyrosine and methionine are metabolised at moderate rates while lysine, serine, threonine, cysteine, glycine, histidine, tryptophan, DOPA and GABA appear to be inert in this respect. Body parts mimic the whole worm in the pattern of transamination of various amino acids with the exception of methionine. Alanine, aspartate and glutamate may transfer their amino group also to pyruvate and oxaloacetate. Alanine and aspartate: 2-oxoglutarate transaminases are located mainly in the cytosol and mitochondrial fractions.     

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

After incubation of muscle preparations with [U-14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. With liver homogenates the degradation of both 2-oxo acids was more complete. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. The degradation of valine was markedly limited by the release of these metabolites. Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO2 and glutamine and glutamate. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. Radioactivity was never found in lactate, pyruvate or alanine. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.     

Role of 5SrRNA as a positive effector of some aminoacyl-tRNA synthetases in macromolecular complexes, with specific reference to methionyl-tRNA synthetase.

1) Rat liver 5SrRNA enhanced the activity of methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex (Fraction B) purified from a rat liver supernatant. 5SrRNA-L5 protein complexes (5SrRNP) had similar effects, whereas other ribosomal RNAs and E. coli 5SrRNA had no effect. 2) 5SrRNA increased the activity of the complex for methionine-dependent ATP-PPi exchange. 3) 5SrRNA increased the activities of methionyl-, arginyl-, and isoleucyl-tRNA synthetases in the complex, but scarcely affected its leucyl-, lysyl-, and glutamyl-tRNA synthetase activities. 4) 5SrRNA increased the activities of the rat liver supernatant for the attachment of [35S]methionine, [3H]isoleucine, [3H]lysine, [3H]proline, [3H]threonine, [3H]tyrosine, and [3H]phenylalanine to endogenous tRNA markedly, and those for [3H]leucine, [3H]arginine, [3H]aspartic acid, and [3H]histidine slightly, but did not affect those for [3H]glutamic acid, [3H]glycine, [3H]valine, [3H]alanine, and [3H]tryptophan. 5) Preincubation of the rat liver supernatant with an antibody against Artemia salina ribosomal protein L5, that cross-reacted with the rat liver ribosomal protein L5, decreased the attachment of [35S]methionine and [3H]isoleucine to endogenous tRNA, and 5SrRNA and 5SRNP enhanced these activities of the supernatant preincubated with antibody. On the other hand, the antibody did not affect that for [3H]alanine. Immune dot blot analysis using the antibody against L5 showed the presence of immunologically the same protein as L5 in the liver supernatant. Northern blot analysis of RNA in the immunoprecipitate prepared from the liver supernatant incubated with the antibody against L5 indicated that 5SrRNA was complexed with L5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compartmentalization of amino acids in surfactant aggregates

Cationic amino acids, arginine and lysine partition differentially from water into aqueous micellar sodium dodecanoate. Conversely, partitioning of serine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, valine, leucine, phenylalanine and isoleucine do not vary appreciably. Partitioning from neat hexane into dodecylammonium propionate trapped water in hexane is, however, dependent upon both electrostatic and hydrophobic interactions. These results imply that the interior of dedecylammonium propionate aggregates is negatively charged and is capable of hydrogen bonding in addition to providing a hydrophobic enviroment. The solubilities of amino acids in neat hexane substantiate the previously derived amino acid hydrophobicity scale. Relevance of partitioning in these systems to the postulated selective amino acid compartmentalization is discussed.     

Utilization of amino acids in growing kidney proximal tubule cell cultures

The growth of rat kidney proximal tubule cells was monitored continuously by the cellular incorporation of [methyl-(14)C] thymidine using scintillating microplates. The radioisotope had no effect on cell proliferation over a 5 day period, neither was it extensively converted to thymine. Leibovitz L-15 medium supplemented with bicarbonate proved a good growth medium and its high levels of carbohydrates and amino acids facilitated the appearance of intermediates in the cells' metabolism of additional radioactive amino acids. Kidney proximal tubule cells had a greater potential to process amino acids than BHK-21 cells. The utilization of amino acids by proximal tubule cells differed from that of other organs. The amino acids could be classified into three classes. Members of the first type were only used for protein synthesis (arginine, lysine, histidine and tyrosine). The second class of amino acids yielded only one or two metabolites (leucine and isoleucine), while the last type gave more than two metabolites (alanine, aspartate, glycine, methionine, proline and valine).     

Incorporation of leucine into phospholipids of Bacteroides thetaiotaomicron.

L-[4,5-3H]- or L-[U-14C]leucine was incorporated by Bacteroides thetaiotaomicron into acid-precipitable material even when the bacteria were treated with concentrations of tetracycline high enough to prevent growth. Similar results were obtained when L-[2,3,4-3H]valine or L-[4,5-3H]isoleucine was used instead of leucine. In bacteria which had been treated with tetracycline, the acid-precipitable label was not solubilized by treatment with protease, lysozyme, or deoxyribonuclease. However, virtually all of the label was extractable with chloroform-methanol, indicating that the label had been incorporated into membrane lipids. Since L-[1-14C]leucine was not incorporated into lipids, leucine was probably decarboxylated before incorporation. When a chloroform extract from bacteria which had been labeled with both [32P]phosphate and [3H]leucine was resolved into component phospholipids by two-dimensional thin-layer chromatography, 3H was incorporated into all of the phospholipids. When these phospholipids were deacylated, the 3H from leucine was associated with released fatty acids rather than with the head groups. Thus, it appears that B. thetaiotaomicron can utilize leucine and similar amino acids not only by incorporating them into protein but also by incorporating portions of these amino acids into membrane phospholipids.     

Evidence for two distinct effector-binding sites in threonine deaminase by site-directed mutagenesis, kinetic, and binding experiments

A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase [Schuller, D. J., Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2, 69-76] and the regulatory domain of E. coli threonine deaminase [Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D. , and Eisenstein, E. (1998) Structure 6, 465-475] led us to make the hypothesis that threonine deaminase could have two binding sites per monomer. To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase. Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A. thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543. Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543. Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme. Y449 interacts with both regulators, isoleucine and valine. However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition.     

BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum

Halophilic archaea, such as eubacteria, use methyl-accepting chemotaxis proteins (MCPs) to sense their environment. We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids. The C-terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation. Hydropathy analysis predicts an enterobacterial-type transducer protein topology for BasT, with an extracellular putative ligand-binding domain flanked by two transmembrane helices and a cytoplasmic domain. BasT-inactivated mutant cells are missing a membrane protein radiolabelled with L-[methyl-3H]-methionine in wild-type cells, confirming that BasT is methylatable and membrane bound. Behavioural analysis of the basT mutant cells by capillary and chemical-in-plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine. The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover. Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine. Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H. salinarum.