Thermosensing ability of Trg and Tap chemoreceptors in Escherichia coli.

The thermosensing ability of the Trg and Tap chemoreceptors in Escherichia coli was investigated after amplifying these receptors in a host strain lacking all four known chemoreceptors (Tar, Tsr, Trg, and Tap). Cells with an increased amount of either Trg or Tap showed mostly smooth swimming and no response to thermal stimuli. However, when the smooth-swimming bias of the cells was reduced by adding Trg- or Tap-mediated repellents, the cells showed clear changes in the swimming pattern upon temperature changes; Trg-containing cells showed tumbling at 23 degrees C but mostly smooth swimming at 32 degrees C, while Tap-containing cells showed smooth swimming at 20 degrees C but tumbling at 32 degrees C. These results indicate that although both Trg and Tap have the ability to sense thermal stimuli, Trg functions as a warm receptor, as reported previously for Tar and Tsr, while Tap functions as a cold receptor.     

Clustering requires modified methyl-accepting sites in low-abundance but not high-abundance chemoreceptors of Escherichia coli

Chemotaxis signalling complexes of Escherichia coli, composed of chemoreceptors, CheA and CheW, form clusters located predominantly at cell poles. As the only kind of receptor in a cell, high-abundance receptors are polar and clustered whereas low-abundance chemoreceptors are polar but largely unclustered. We found that clustering was a function of the cytoplasmic, carboxyl-terminal domain and that effective clustering was conferred on low-abundance receptors by addition of the approximately 20-residue sequence from the carboxyl terminus of either high-abundance receptor. These sequences are different but share a carboxyl-terminal pentapeptide that enhances adaptational covalent modification and allows a physiological balance between modified and unmodified methyl-accepting sites, implying that receptor modification might influence clustering. Thus we investigated directly effects of modification state on chemoreceptor clustering. As the sole receptor type in a cell, low-abundance receptors were clustered only if modified, but high-abundance receptors were clustered independent of extent of modification. This difference could mean that the two receptor types are fundamentally different or that they are poised at different positions in the same conformational equilibrium. Notably, no receptor perturbation we tested altered a predominant location at cell poles, emphasizing a distinction between determinants of clustering and polar localization.     

Primary structure and properties of an inactive mutant aspartate transcarbamoylase.

A mutant form of Escherichia coli aspartate transcarbamoylase (ATCase) which lacks catalytic activity has been purified and characterized (Wall, K.A., Flatgaard, J.E., Gibbons, I., and Schachman, H.K. (1979) J. Biol. Chem 254, 11910-11916). Peptide mapping of the mutant and wild type catalytic chains followed by the determination of the amino acid sequence of the one altered peptide in the mutant indicated that a glycyl residue was replaced by aspartic acid. This substitution is located at position 125 in the tentative sequence kindly provided by W. Konigsberg (personal communication). The mutant protein has an overall secondary structure similar to that of the wild type as indicated by circular dichroism spectroscopy. However, marked changes in the reactivity of several amino acid residues were demonstrated. Lysyl residue 84 which in the wild type subunits reacts specifically with pyridoxal 5'-phosphate is only slightly reactive in the mutant even though the peptide containing that residue was not altered in amino acid composition. Another residue, cysteinyl 46, which is thought to be in the active site, is much more reactive toward p-hydroxymercuribenzoate in the mutant subunit than in the wild type protein. Finally, tyrosyl residue 213, which according to recent crystallographic studies is not near the active site and which exhibits an unusually low pK (9.1) in the wild type catalytic subunits, appears to have its pK shifted to 10.5 or higher as a result of the mutation. The evidence indicates that the substitution of an aspartyl for a glycyl residue at a region of the amino acid sequence remote from those residues in the active site causes sufficient modification of the tertiary structure to cause the loss of enzyme activity and to affect the reactivity of other residues in the protein. Moreover, the quaternary structure of the intact enzyme is altered as well since the subunit interactions are greatly weakened.     

A cis-proline to alanine mutant of E. coli aspartate transcarbamoylase: kinetic studies and three-dimensional crystal structures

The only cis-proline residue in Escherichia coli aspartate transcarbamoylase has been replaced by alanine using site-specific mutagenesis. The Pro268-->Ala enzyme exhibits a 40-fold reduction in enzyme activity and decreased substrate affinity toward carbamoyl phosphate and aspartate compared to the corresponding values for the wild-type enzyme. The concentration of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) required to activate the mutant enzyme to the same extent as the wild-type enzyme is significantly increased. The heterotropic effects of ATP and CTP upon the Pro268-->Ala enzyme are also altered. Crystal structures of the Pro268-->Ala enzyme in both T- and R-states show that the cis-peptidyl linkage between Leu267 and Ala268 is maintained. However, the tertiary structure of both the catalytic and regulatory chains has been altered by the amino acid substitution, and the mobility of the active-site residues is increased for the R-state structure of Pro268-->Ala enzyme as comparison with the wild-type R-state structure. These structural changes are responsible for the loss of enzyme activity. Thus, Pro268 is required for the proper positioning of catalytically critical residues in the active site and is important for the formation of the high-activity high-affinity R-state of E. coli aspartate transcarbamoylase.     

Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.

A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

The methyl-accepting chemotaxis proteins of Escherichia coli. Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I

The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents. The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels. We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides. At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times. The second methyl-accepting tryptic peptide contains arginine and may be methylated twice. Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times. The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells. The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.     

Thermosensing properties of Escherichia coli tsr mutants defective in serine chemoreception.

Tsr, a chemoreceptor for serine and repellents in Escherichia coli, also functions as a thermoreceptor. The relationship between the chemoreceptor and thermoreceptor functions of Tsr was examined in five tsr mutants with altered serine detection thresholds. The thermosensing abilities of the mutant Tsr proteins were not affected by the alterations in their affinities to serine. In contrast, the ability of serine to inactivate thermoreceptor function was altered in these mutants. The minimal serine concentration required for thermoreceptor inactivation was directly related to the decreased affinity of the mutant Tsr for serine. The amino acid replacements in the mutant receptors were deduced from DNA sequence analyses and occurred at two different locations in the presumed periplasmic domain of Tsr. Two mutations caused histidine or cysteine replacements at arginine 64, whereas three others caused isoleucine or proline replacements at threonine 156.     

Platelet aggregation inhibitory activity of mutant proinsulin with C-peptide replaced by CRGDSC sequence

A CRGDSC peptide was introduced into an inactive human proinsulin molecule between the B30 site and A1 site to replace the C-peptide. The constructed RGD-proinsulin gene was overexpressed in E. coli and the protein purified. It showed an inhibitory activity of platelet aggregation with an IC₅₀ value of 0.35 M, while native insulin and proinsulin as controls did not exhibit any inhibitory activity. Meanwhile, the RGD-proinsulin demonstrated only 0.05% of insulin receptor binding activity and almost no insulin activity in in vivo assay.     

Component of the Rhodospirillum centenum photosensory apparatus with structural and functional similarity to methyl-accepting chemotaxis protein chemoreceptors

Photosynthetic bacteria respond to alterations in light conditions by migrating to locations that allows optimal use of light as an energy source. Studies have indicated that photosynthesis-driven electron transport functions as an attractant signal for motility among purple photosynthetic bacteria. However, it is unclear just how the motility-based signal transduction system monitors electron flow through photosynthesis-driven electron transport. Recently, we have demonstrated that the purple photosynthetic bacterium Rhodospirillum centenum is capable of rapidly moving swarm cell colonies toward infrared light as well as away from visible light. Light-driven colony motility of R. centenum has allowed us to perform genetic dissection of the signaling pathway that affects photosynthesis-driven motility. In this study, we have undertaken sequence and mutational analyses of one of the components of a signal transduction pathway, Ptr, which appears responsible for transmitting a signal from the photosynthesis-driven electron transport chain to the chemotaxis signal transduction cascade. Mutational analysis demonstrates that cells disrupted for ptr are defective in altering motility in response to light, as well as defective in light-dependent release of methanol. We present a model which proposes that Ptr senses the redox state of a component in the photosynthetic cyclic electron transport chain and that Ptr is responsible for transmitting a signal to the chemotaxis machinery to induce a photosynthesis-dependent motility response.     

Equilibrium unfolding of mutant apomyoglobins with substitutions of conserved nonfunctional residues by alanine

The problems of protein aggregation and protein misfolding in the cell are connected with the appearance of many genetic diseases. Both processes can be a consequence of substitutions of certain amino acid residues in proteins. The substitutions can influence the protein stability and protein folding rates in both the intermediate and the native states. We have studied equilibrium urea unfolding of mutant forms of apomyoglobin with substitutions of conserved nonfunctional residues by Ala to estimate their influence on protein stability. These residues include Val10, Trp14, Ilel11, Leu115, Met131 and Leu135. Conformational transitions were monitored by intrinsic Trp fluorescence and by circular dichroism spectra in the far UV region. Free energy changes upon the transition from the native to intermediate state and from the intermediate to unfolded state were determined. It was shown that all substitutions used lead to an appreciable decrease of the apomyoglobin native state stability, whereas the stability of the intermediate state is affected substantially smaller.     

Increased oxygen affinity for hemoglobin Sawara: alphaA4(6) aspartic acid replaced by alanine.

The oxygen binding property of Hb Sawara (alphaA4 Asp replaced by Ala) was studied at different pH values with and without addition of 2,3-diphosphoglycerate. The oxygen affinity of Hb Sawara was shown to be increased, the difference of the log P50 value between normal and abnormal hemoglobins being 0.37 at pH 7.0. Both the magnitude of the alkaline Bohr effect and the effect of 2,3-diphosphoglycerate upon oxygen affinity of Hb Sawara were comparable to those of Hb A. The amino acid substitution of alanine for alphaA4 aspartic acid might result in the loss of a stabilizing force for ionic interaction between the alpha-amino group of NA (1)alpha1 valine and the alpha-carboxyl of HC3(141)alpha2 arginine in the deoxy-form.     

Effects of glutamines and glutamates at sites of covalent modification of a methyl-accepting transducer.

Chemotactic transducer proteins of Escherichia coli contain four or five methyl-accepting glutamates that are crucial for sensory adaptation and gradient sensing. Two residues arise from posttranslational deamidation of glutamines to yield methyl-accepting glutamates. We addressed the significance of this arrangement by creating two mutated trg genes: trg(5E), coding for a transducer in which all five modification sites were synthesized as glutamates, and trg(5Q), in which all five were glutamines. We found that the normal (3E,2Q) configuration was not an absolute requirement for synthesis, assembly, or stable maintenance of transducers. Both mutant proteins were methylated, although Trg(5Q) had a reduced number of methyl-accepting sites because two glutamines at adjacent residues were blocked for deamidation and thus could not become methyl-accepting glutamates. The glutamine-glutamate balance had striking effects on signaling state. Trg(5E) was in a strong counterclockwise signaling configuration, and Trg(5Q) was in a strong clockwise signaling induced by ligand binding, and alanines substituted at modification sites had an intermediate effect. Chemotactic migration by growing cells containing trg(5E) or trg(5Q) exhibited reduced effectiveness, probably reflecting perturbations of the counterclockwise/clockwise ratio caused by newly synthesized transducers not modified rapidly enough to produce a balanced signaling state during growth. These defects were evident for cells in which other transducers were not available to contribute to balanced signaling or were present at lower levels than the mutant proteins.     

Acetylcholinesterase in singly and multiply innervated muscles of normal and dystrophic chickens. II. Effects of denervation.

Neural regulation of mature normal fast twitch muscle of the chicken suppresses high activity, extrajunctional localization, and isozyme forms of acetylcholinesterase (AChE) characteristic of embryonic, denervated and dystrophic muscle. Normal adult slow tonic muscle ofthe chicken retains intermediate levels of activity and embryonic isozyme forms but not extrajunctional activity; it is not affected by muscular dystrophy. The hypothesis that neural regulation of the AChE system is lacking in slow tonic muscle and thus not affected by dystrophy was tested by denervating the fast twitch posterior latissimus dorsi and slow tonic anterior latissimus dorsi muscles of normal and dystrophic chickens. Extrajunctional AChE activity and embryonic isozyme forms increased, then declined, in both muscles. The results suggest that ocntrol of AChE is qualitatively similar in slow tonic and fast twitch muscle of the chicken.     

Kinetics of receptor modification. The multiply methylated aspartate receptors involved in bacterial chemotaxis

A method for determining the extent of methyl esterification of each of the four potential sites on the aspartate receptors involved in chemotaxis in Escherichia coli and Salmonella typhimurium is presented. In this procedure, radioactive methyl esters are incorporated into the receptors, the receptors are cleaved by trypsin and the V8 protease from Staphylococcus aureus, and the four fragments containing sites of methylation are separated by high performance liquid chromatography. Using this technique, we find that the rate of methyl esterification increases at all four sites after stimulation with the "attractant" aspartate, suggesting that all four sites of modification are involved in adaptation to aspartate. We also find that the rate of methyl esterification at each site is correlated with the homology between the protein sequence at that site and the "consensus" sequence, Glu-Glu-X-X-Ala-Thr/Ser.     

Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement.

Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined. Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH. Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant. The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant. No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme. However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type. NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes. Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases. This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex.     

Insulin analogs with B24 or B25 phenylalanine replaced by biphenylalanine

B24 and B25 phenylalanines （Phe） play important roles in insulin structure and function. Insulin analogs with B24 Phe or B25 Phe replaced by biphenylalanine （Bip） were prepared by enzymatic semisynthesis. The biological activities were determined by receptor binding assay and in vivo mouse convulsion assay. The results showed that B25 Bip insulin has 139% receptor binding activity and 50% in vivo biological activity, whereas B24 Bip insulin is inactive, when compared with native insulin, suggesting that B24 Phe is crucial for insulin activity. The structures in solution were studied by circular dichroism and fluoremetry, and our results suggested that the insulin analogs with low activities tend to be more tightly packed. The association properties were studied by size exclusion chromatography. The Bip.amide replacement of B24 Phe in deshexapeptide insulin or B25 Phe in despentapeptide insulin will cause the monomeric B24 Phe-amide deshexapeptide insulin or B25 Phe-amide despentapeptide insulin to associate and form dimers, whereas the mutations of B24 Phe in insulin will make insulin dimers dissociate into insulin monomers.     

Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.     

Discrimination of DNA binding sites by mutant p53 proteins.

Critical determinants of DNA recognition by p53 have been identified by a molecular genetic approach. The wild-type human p53 fragment containing amino acids 71 to 330 (p53(71-330)) was used for in vitro DNA binding assays, and full-length human p53 was used for transactivation assays with Saccharomyces cerevisiae. First, we defined the DNA binding specificity of the wild-type p53 fragment by using systematically altered forms of a known consensus DNA site. This refinement indicates that p53 binds with high affinity to two repeats of PuGPuCA.TGPyCPy, a further refinement of an earlier defined consensus half site PuPuPuC(A/T).(T/A) GPyPyPy. These results were further confirmed by transactivation assays of yeast by using full-length human p53 and systematically altered DNA sites. Dimers of the pentamer AGGCA oriented either head-to-head or tail-to-tail bound efficiently, but transactivation was facilitated only through head-to-head dimers. To determine the origins of specificity in DNA binding by p53, we identified mutations that lead to altered specificities of DNA binding. Single-amino-acid substitutions were made at several positions within the DNA binding domain of p53, and this set of p53 point mutants were tested with DNA site variants for DNA binding. DNA binding analyses showed that the mutants Lys-120 to Asn, Cys-277 to Gln or Arg, and Arg-283 to Gln bind to sites with noncanonical base pair changes at positions 2, 3, and 1 in the pentamer (PuGPuCA), respectively. Thus, we implicate these residues in amino acid-base pair contacts. Interestingly, mutant Cys-277 to Gln bound a consensus site as two and four monomers, as opposed to the wild-type p53 fragment, which invariably binds this site as four monomers.     

The regulation of alanine and aspartate aminotransferase by different aminothiols and by vitamin B-6 derivatives

We examined the effects on alanine aminotransferase and aspartate aminotransferase of different aminothiols (l-cysteine, d-cysteine, cysteamine, l-cysteine ethyl ester, l-cysteine methyl ester) and several vitamin B-6 derivatives (pyridoxal, pyridoxamine, pyridoxol, pyridoxol 5′-phosphate), before and after treatment with KOCN, which transforms these molecules into the corresponding carbamoyl derivatives. Only GTP, and not GOT, was specifically inhibited by l-cysteine and, to a lesser extent, by d-cysteine. The association reaction: PLP + apo GPT ↔ holo GPT was inhibited by the vitamin B-6 derivatives, and this inhibition was prevented by pretreatment of the vitamin B-6 derivatives with KOCN. All the observed effects occurred at pH 7, 37°C, at mM and even lower concentrations of reagents. Hence, they all potentially play a physiological role, in the regulation of the PLP dependent enzymes and of the vitamin B-6 levels in the cell.