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PubMed2005

Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding.

Staphylococcal nuclease is a single domain protein with 149 amino acids. It has no disulfide bonds, which makes it a simple model for the study of protein folding. In this study, 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in thermal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lower DeltaH(cal) values than the wild-type protein. Furthermore, two mutants, E75Q and E129Q, were created and the results imply that substitution of the Gly residue has little influence on destabilization of the secondary structure that leads to the large perturbation of the tertiary protein structure stability. Two local stable areas formed by the charge-charge interactions around E75 and E129 with particular positively charged amino acids are thus identified as being significant in maintenance of the three-dimensional structure of the protein.

Aspartic Acid↗
PubMed2003

TRPC1 is required for functional store-operated Ca2+ channels. Role of acidic amino acid residues in the S5-S6 region.

The exact role of TRPC1 in store-operated calcium influx channel (SOCC) function is not known. We have examined the effect of overexpression of full-length TRPC1, depletion of endogenous TRPC1, and expression of TRPC1 in which the proposed pore region (S5-S6, amino acids (aa) 557-620) was deleted or modified by site-directed mutagenesis on thapsigargin- and carbachol-stimulated SOCC activity in HSG cells. TRPC1 overexpression induced channel activity that was indistinguishable from the endogenous SOCC activity. Transfection with antisense hTRPC1 decreased SOCC activity although characteristics of SOCC-mediated current, I(SOC), were not altered. Expression of TRPC1 Delta 567-793, but not TRPC1 Delta 664-793, induced a similar decrease in SOCC activity. Furthermore, TRPC1 Delta 567-793 was co-immunoprecipitated with endogenous TRPC1. Simultaneous substitutions of seven acidic aa in the S5-S6 region (Asp --> Asn and Glu --> Gln) decreased SOCC-mediated Ca(2+), but not Na(+), current and induced a left shift in E(rev). Similar effects were induced by E576K or D581K, but not D581N or E615K, substitution. Furthermore, expressed TRPC1 proteins interacted with each other. Together, these data demonstrate that TRPC1 is required for generation of functional SOCC in HSG cells. We suggest that TRPC1 monomers co-assemble to form SOCC and that specific acidic aa residues in the proposed pore region of TRPC1 contribute to Ca(2+) influx.

Amino Acid Sequence↗
PubMed1997

Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

Amino Acid Sequence↗
PubMed1996

Mutations at two conserved acidic amino acids in the glycoprotein of vesicular stomatitis virus affect pH-dependent conformational changes and reduce the pH threshold for membrane fusion.

Recently, we had shown that amino acid substitutions at residues 124, 127, and 133 either abolished or drastically altered the fusion activity of the glycoprotein (G protein) of vesicular stomatitis virus (VSV), indicating that this region is important for membrane fusion and may constitute an internal fusion domain. In this report we show that amino acid substitutions at two conserved acidic residues located at the C-terminal end of the putative fusion domain also affect the fusion activity of G protein. Two substitutions, D127-L and E139-L, slightly reduced the pH threshold at which G protein mediates membrane fusion. However, both D137-L and E139-L had fusion activities equivalent to that of wild-type G protein after exposure to a pH of 5.7 or below. To determine if the fusion activity of G protein required an acidic residue in this region both D137 and E139 were replaced with serine. This double substitution also reduced the pH threshold for fusion activity, but the effect appeared to be less severe than that for either of the single substitutions. The mutated G protein cDNAs were also introduced into a VSV minigenome to examine the effect of the substitutions on virus assembly and infectivity. All three mutant G proteins assembled into particles and these virions were infectious despite the altered fusion activities of the mutant G proteins. Although particles containing the mutant proteins were infectious they appeared to be attenuated, suggesting that these two acidic residues, which are conserved in several distantly related rhabdoviruses, play an important role in maintaining virus fitness.

Animals↗
PubMed1993

The anomalous electrophoretic behavior of the human papillomavirus type 16 E7 protein is due to the high content of acidic amino acid residues.

In the presence of sodium dodecyl sulfate and 2-mercaptoethanol, the human papillomavirus 16 E7 protein migrates as a 17 kD protein during polyacrylamide gel electrophoresis. However, the theoretical molecular mass of this protein is approximately 11 kD. Substitution of 2 basic amino acids for 2 acidic residues in the amino terminus of the protein restored normal electrophoretic mobility. Furthermore, neutralization of negative charge through chemical modification of the wild type protein normalized migration. These results indicate that the substantial net negative charge of the wild type E7 protein is responsible for its anomalous electrophoretic behavior.

Amino Acid Sequence↗
PubMed1966

Utilization in vivo of glucose and volatile fatty acids by sheep brain for the synthesis of acidic amino acids.

1. Free glutamic acid, aspartic acid, glutamic acid from glutamine and, in some instances, the glutamic acid from glutathione and the aspartic acid from N-acetyl-aspartic acid were isolated from the brains of sheep and assayed for radioactivity after intravenous injection of [2-(14)C]glucose, [1-(14)C]acetate, [1-(14)C]butyrate or [2-(14)C]propionate. These brain components were also isolated and analysed from rats that had been given [2-(14)C]propionate. The results indicate that, as in rat brain, glucose is by far the best precursor of the free amino acids of sheep brain. 2. Degradation of the glutamate of brain yielded labelling patterns consistent with the proposal that the major route of pyruvate metabolism in brain is via acetyl-CoA, and that the short-chain fatty acids enter the brain without prior metabolism by other tissue and are metabolized in brain via the tricarboxylic acid cycle. 3. When labelled glucose was used as a precursor, glutamate always had a higher specific activity than glutamine; when labelled fatty acids were used, the reverse was true. These findings add support and complexity to the concept of the metabolic ;compartmentation' of the free amino acids of brain. 4. The results from experiments with labelled propionate strongly suggest that brain metabolizes propionate via succinate and that this metabolic route may be a limited but important source of dicarboxylic acids in the brain.

Journal Article↗
PubMed1997

Family of neutral and acidic amino acid transporters: molecular biology, physiology and medical implications.

Glutamate transporters and structurally related neutral amino acid transporters constitute a distinct family of Na(+)-dependent transporters. The different transporters of this family share similar structural traits, and exhibit different yet comparable functions. Significant recent advances in our understanding of the structure and function of these transporters include: a new twist in our knowledge of ion-coupling stoichiometry; the knockout of glutamate transporters, which reveals a major role for glial glutamate transporters; and new insights into the regulation of glutamate transporter expression.

ATP-Binding Cassette Transporters↗
PubMed2004

Effect of pH on the stability and structure of yeast hexokinase A. Acidic amino acid residues in the cleft region are critical for the opening and the closing of the structure.

pH and salts have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. In this work, calorimetry, CD, and fluorescence studies have been carried out to understand the pH-dependent conformational changes of the two-domain protein yeast hexokinase A. In conjunction with the crystal structural data available, the present results have enabled the complete characterization and analysis of the pH-dependent conformational changes of the enzyme that have strong implications in understanding its structure-function relationship. The calorimetric profiles show a single thermal transition in the acidic pH range, whereas two independent transitions were observed in the alkaline pH range, suggesting the structural merger of the domains at the acidic pH. Comparison of the thermal transitions at pH 8.5 studied by different techniques suggests that the first transition corresponds to the smaller domain, and the second transition corresponds to the larger domain. The acid-denatured state of hexokinase A has high secondary structure content with little or no tertiary interactions and binds to the hydrophobic dye 8-anilinonaphthalene-1-sulfonic acid, suggesting that it is a molten globule-like state, whereas the alkali-denatured state is less structured than the acid-denatured state but more structured than the urea-denatured state, suggestive of a premolten globule-like state. Structural analysis using the published hexokinase B structure as well as the hexokinase A structure with the revised amino acid sequence in conjunction with the results obtained by us suggests that the ionization state of the acidic residues at the active site could regulate domain movements that are responsible for the opening and the closure of the cleft between the two domains and in turn affect the structure and function of the enzyme.

Acids↗
PubMed1999

Structure and function of metal chelators produced by plants: the case for organic acids, amino acids, phytin, and metallothioneins.

Plants produce a range of ligands for cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn). Cd- and Zn-citrate complexes are prevalent in leaves, even though malate is more abundant. In the xylem sap moving from roots to leaves, citrate and histidine are the principal ligands for Cu, Ni, and Zn. Phosphorus-rich globular bodies in young roots are probably Zn-phytate. Metallothioneins (MTs) are cysteine (Cys)-rich ligands. Plants produce class II MTs (MT-IIs) which differ from the archetypal mammalian MT-I in the location and number of Cys. The Ec protein from wheat embryos has Cys in three domains, binds Zn, and disappears with seedling development. The first 59 amino acids have been sequenced for the protein. Fifty-eight genes for MT-IIs, from a range of plants and tissues, predict proteins with Cys in two domains. Most of the predicted proteins have not been isolated, and their metal binding is poorly documented. Three protein bands, corresponding to six MT genes, have been isolated from Arabidopsis, and the amino acids sequenced for nine fragments. The MT-IIIs are atypical, nontranslationally synthesized polypeptides with variously repeating gamma-glutamylcysteine units. Of the five families known, those with carboxy-terminal glycine are the most widespread among plants, algae, and certain yeasts. A heterogeneous grouping of these molecules form Cd-binding complexes with tetrahedral coordination and a Cd-sulfur interatomic distance of 2.52 A. One complex is cytosolic, the dominant one is vacuolar. Together, they can bind a large proportion of cellular Cd; other ligands may also function. Little is known about the counterpart situation for Cu and Zn.

Cadmium↗
PubMed2004

Functional analysis of acidic amino acids in the cytosolic tail of the Na+/H+ exchanger.

The mammalian Na(+)/H(+) exchanger is a membrane protein with a C-terminal regulatory cytosolic domain and an N-terminal membrane domain. Na(+)/H(+) exchanger isoform 1 (NHE1) possesses a conserved amino acid sequence of seven consecutive acidic residues in the distal region of the cytosolic tail. We examined the structural and functional role of this acidic sequence. In human NHE1, varying mutations of the sequence (753)EEDEDDD(759) resulted in defective NHE1 activity. Mutation of the core acid sequence, (755)DED(757), or of the entire sequence caused a decrease in the activity of NHE1 in response to acute acid load. This was not due to changes in Na(+) affinity but rather due to decreased maximum velocity of the protein and delayed activation. Mutation of the target sequence did not affect the ability of the cytoplasmic domain to bind carbonic anhydrase II or tescalcin but did affect calmodulin binding. Mutation of the acidic domain also caused altered sensitivity to trypsin and changes in size of the protein in gel-filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results demonstrate that the acidic sequence is critical in maintaining proper conformation of the cytosolic domain, calmodulin binding, and in maintenance of Na(+)/H(+) exchanger activity.

Amino Acid Sequence↗
PubMed2006

Several acidic amino acids in the N-domain of insulin-like growth factor-binding protein-5 are important for its transactivation activity.

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of cultured cells and has transactivation activity. Here we report the nuclear localization of endogenous IGFBP-5 in mouse embryonic skeletal cells. Chromatin immunoprecipitation experiments indicated that IGFBP-5 interacts with the nuclear histone-DNA complex. Using a series of deletion mutants, the transactivation domain of IGFBP-5 was mapped to its N-terminal region. Intriguingly, the transactivation activity of IGFBP-5 is masked by negative regulatory elements located in the L- and C-domains. Among the other IGFBPs, the N-domains of IGFBP-2 and -3 also had strong transactivation activity, whereas those of IGFBP-1 and -6 had no activity. The IGFBP-4 N-domain had modest activity. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (e.g. E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (e.g. Q56R) significantly reduced transactivation activity. The E8A/D11S/E12A triple mutant and E52A/Q56R double mutants showed further reduced activity. The combinatory mutants had essentially no transactivation activity. Taken together, our results indicate that there are several conserved residues in the IGFBP-5 N-terminal region that are critical for transactivation and that IGFBP-2 and -3 also have strong transactivation activity in their N-domains.

Amino Acid Sequence↗
PubMed2005

Cholic-acid-based fluorescent sensor for dicarboxylates and acidic amino acids in aqueous solutions.

[structure: see text] The binding affinities of a cholic-acid-based fluorescent neutral receptor toward dicarboxylate anions and amino acids have been investigated in a CH3OH/H2O system (1:1, 0.01 M HEPES buffer, pH = 7.4) by fluorescence titration experiments. The synthetic host bearing four convergent functionalities strongly binds glutamate via multiple hydrogen bonds with a binding constant of (5.57 +/- 0.88) x 10(6).

Amino Acids, Acidic↗
PubMed1998

Negative ion postsource decay time-of-flight mass spectrometry of peptides containing acidic amino acid residues.

Acidic peptides have been studied by negative ion postsource decay (PSD) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The peptides contained from 5 to 16 residues and were chosen on the basis of their patterns of the acidic residues. Using typical MALDI sample preparation techniques employing an acidic matrix, gastrin I (1-14), and epidermal mitosis inhibiting pentapeptide yielded much larger deprotonated ion signals, [M - H]-, than protonated ions, [M + H]+. This may be due to their absence of basic residues, coupled with their arrays of acidic residues. The PSD fragmentation of the peptide negative ions showed that an array of acidic residues, as in gastrin I (1-14), yielded simple spectra containing mainly backbone cleavage ions from the C-terminus. Hirudin (54-65), which contains two sets of two consecutive Glu residues, and fibrinopeptide A and fibrinopeptide B, with isolated acidic residues, also showed backbone cleavages as common fragment ions. In addition, the two sets of isolated consecutive amino acid residues in Cys(Bzl)84-CD4 (81-92) and hirudin (54-56) yielded internal ions from the cleavages at the (O=C)-NH bond between the acidic residues. Also observed were ions with unique side chain losses, such as the loss of C6H4O from a tyrosine residue and SCH2C6H5 and CH2C6H5 from a benzylated cysteine residue. Compared to the positive mode, the negative-ion PSD yielded fewer fragments which usually involved only one type of backbone cleavage (e.g., [Yn - H2O]-). These simple spectra aided interpretation. Overall, the acidic peptides studied yielded negative ion PSD spectra that were useful for peptide sequencing.

Amino Acids↗
PubMed2004

A dileucine motif and a cluster of acidic amino acids in the second cytoplasmic domain of the batten disease-related CLN3 protein are required for efficient lysosomal targeting.

The juvenile form of ceroid lipofuscinosis (Batten disease) is a neurodegenerative lysosomal storage disorder caused by mutations in the CLN3 gene. CLN3 encodes a multimembrane-spanning protein of unknown function, which is mainly localized in lysosomes in non-neuronal cells and in endosomes in neuronal cells. For this study we constructed chimeric proteins of three CLN3 cytoplasmic domains fused to the lumenal and transmembrane domains of the reporter proteins LAMP-1 and lysosomal acid phosphatase to identify lysosomal targeting motifs and to determine the intracellular transport and subcellular localization of the chimera in transfected cell lines. We report that a novel type of dileucine-based sorting motif, EEEX(8)LI, present in the second cytoplasmic domain of CLN3, is sufficient for proper targeting to lysosomes. The first cytoplasmic domain of CLN3 and the mutation of the dileucine motif resulted in a partial missorting of chimeric proteins to the plasma membrane. At equilibrium, 4-13% of the different chimera are present at the cell surface. Analysis of lysosome-specific proteolytic processing revealed that lysosomal acid phosphatase chimera containing the second cytoplasmic domain of CLN3 showed the highest rate of lysosomal delivery, whereas the C terminus of CLN3 was found to be less efficient in lysosomal targeting. However, none of these cytosolic CLN3 domains was able to interact with AP-1, AP-3, or GGA3 adaptor complexes. These data revealed that lysosomal sorting motifs located in an intramolecular cytoplasmic domain of a multimembrane-spanning protein have different structural requirements for adaptor binding than sorting signals found in the C-terminal cytoplasmic domains of single- or dual-spanning lysosomal membrane proteins.

Acid Phosphatase↗
PubMed1981

The postexcitatory effects of acidic amino acids on spinal neurones.

The excitation of some neurones in the spinal cord of rats by L-glutamate and L-aspartate is followed by a period of reduced excitability. This effect is not observed after excitation by D-glutamate nor, in the case of Renshaw cells, by acetylcholine. The depressions following L-glutamate were reduced by bicuculline and those after aspartate by strychnine, suggesting that they may have been caused through decarboxylation of the excitatory amino acids to yield the inhibitory compounds gamma-aminobutyric acid and beta-alanine, respectively.

Action Potentials↗
PubMed1989

Effect of tunicamycin, an inhibitor of protein glycosylation, on the high-affinity transport of acidic amino acid neurotransmitters in C6 glioma cells.

The effect of tunicamycin, an inhibitor of protein glycosylation, on the high-affinity transport of D-aspartate was investigated in C6 astrocytoma cells. A concentration of tunicamycin (1 microgram/ml) that after 24 h exposure inhibited the rate of transport by 70% and incorporation of [3H]mannose by 82-95% had only a small effect on [14C]leucine incorporation into protein and cell growth (20% reduction). Tunicamycin decreased the Vmax for transport without affecting the Km, which suggests that inhibition of glycosylation reduces the number of competent transporters on the surface of the plasma membrane. The decrease in the velocity of uptake was attenuated when C6 cells were treated with tunicamycin in the presence of protease inhibitors, indicating that the underglycosylated carriers are subject to enhanced proteolytic degradation. Incubation in drug-free medium following treatment with 1 microgram/ml of tunicamycin for 24 h resulted in recovery of D-aspartate transport within 48 h. This recovery was prevented by the presence of cycloheximide, which indicates that synthesis of new transporters is necessary for the restoration of normal rates of D-aspartate uptake. These results support our earlier postulate that the high-affinity carriers for amino acid transmitters are transmembrane glycoproteins.

Amino Acids↗
PubMed1984

The effect of a keto acid-amino acid supplement to a restricted diet on the progression of chronic renal failure.

We treated 24 patients who had chronic renal failure with a low-phosphorus diet containing 20 to 30 g of mixed-quality protein, supplemented by amino acids and their keto analogues. Seventeen patients had well-defined rates of progression before treatment, as assessed by serial determinations of serum creatinine levels. By extrapolating these rates of progression, we found that 10 of the 17 (59 per cent) had a clinically important slower rise in creatinine levels during long-term treatment (average, 20 months) than predicted; none had a faster rise than predicted. Seven of the 17 patients began treatment before creatinine reached the level of 8 mg per deciliter; in six of the seven, followed for an average of 22 months, creatinine has remained at or below the level at the start of treatment. Nutrition, as assessed by body weight, nitrogen balance, serum albumin, and serum transferrin, has been well maintained. This regimen slowed or arrested the rise in creatinine levels and thus must have slowed or halted the progression of renal insufficiency in a majority of cases, especially when treatment was initiated before creatinine had reached the level of 8 mg per deciliter. The mechanism underlying this effect remains to be determined.

Adult↗