NO news is good news.
A startlingly simple molecule unites neuroscience, physiology, and immunology, and revises scientists' understanding of how cells communicate and defend themselves.
Animals↗
Biomedical subjects
D E Koshland
Publications and source records attributed to D E Koshland.
Structure and dynamics of transmembrane signaling by the Escherichia coli aspartate receptor.
The structure of the cytosolic extension of the first transmembrane region (TM1) of the Escherichia coli aspartate receptor (residues 3, 4, and 5) and conformational changes within that region have been characterized by targeted cross-linking studies and by measurement of the effect of aspartate binding on cross-linking and methylation rates and compared with the periplasmic extension of the same helix. These experiments show that (1) the cytosolic extension of TM1 is helical, with residues 4 and 4' closest together at the dimer interface; (2) the helix is more solvent-exposed at the cytosolic side of the membrane than on the periplasmic side; and (3) aspartate binding enhances the rate of cross-linking at Cys 4, and the resulting cross-linked receptor displays aspartate-induced transmembrane increases in methylation by the cytoplasmic methylase (the CheR protein). We conclude that aspartate induces a conformational change that does not involve large intersubunit movements that lead to an increase in distance between the cytosolic ends of the first membrane-spanning helices; rather, the motion involved is largely contained within individual subunits, possibly resulting in a small movement between positions 4 and 4'.
Aspartic Acid↗
Retention of habituation in PC12 cells.
The time course of habituation and recovery of neurotransmitter release was measured in neuronally differentiated PC12 cells stimulated with either acetylcholine or ATP. The release of norepinephrine in response to either stimulant declined exponentially with repeated presentation of that stimulant. When the stimulus was withheld, the cells' ability to respond recovered to initial levels with an exponential time course. The rate of response recovery depended on the stimulant used and, in the case of stimulation with acetylcholine, on the number of previous stimuli. After habituation and recovery, or partial recovery, of norepinephrine release, the response to a second series of repetitive stimuli declined more rapidly than in the naive case. This increase in habituation rate was dependent on the number of previous stimuli and, in the case of stimulation with acetylcholine, was stable with time for at least 90 min after stimulation. These phenomena are analogous to characteristics of short- and long-term memories of habituative learning observed in behavioral studies. Kinetic equations based on a putative reversible stimulation-dependent inactivation of the cellular response mechanism were used to analyze the rates of habituation and response recovery.
Acetylcholine↗
Regulation of neurosecretory habituation in PC12 cells: parallel pathways used by cAMP and calcium.
Habituation of norepinephrine secretion in PC12 cells serves as a model for understanding the molecular mechanisms of simple memory processes in neurons. Elevation of intracellular cAMP levels by incubation with N-ethylcarboxamidoadenosine (NECA) or forskolin increased norepinephrine secretion in response to depolarization by high potassium or stimulation with acetylcholine. The extent to which cAMP altered norepinephrine secretion was dependent on the timing of its elevation, and it also altered the rate of habituation under certain conditions. However, cAMP increased norepinephrine secretion by a pathway distinct from that governing internal calcium levels, which correlates with habituation in the absence of elevated cAMP. An inverse correlation was found between the ability of calcium to lower NECA-induced cAMP levels and the ability of calcium to cause norepinephrine secretion. A model is proposed in which a single calcium-dependent pathway modulates both norepinephrine secretion and cAMP metabolism.
Acetylcholine↗
Prediction of the structure of a receptor-protein complex using a binary docking method.
To validate procedures of rational drug design, it is important to develop computational methods that predict binding sites between a protein and a ligand molecule. Many small molecules have been tested using such programs, but examination of protein-protein and peptide-protein interactions has been sparse. We were able to test such applications once the structures of both the maltose-binding protein (MBP) and the ligand-binding domain of the aspartate receptor, which binds MBP, became available. Here we predict the binding site of MBP to its receptor using a 'binary docking' technique in which two MBP octapeptide sequences containing mutations that eliminate maltose chemotaxis are independently docked to the receptor. The peptides in the docked solutions superimpose on their original positions in the structure of MBP and allow the formation of an MBP-receptor complex. The consistency of the computational and biological results supports this approach for predicting protein-protein and peptide-protein interactions.
Amino Acid Sequence↗
The microbial wars.
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Bacterial Infections↗
Conflict of interest policy.
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Biomedical Research↗
The fifth Datta Lecture. Structural similarities between the aspartate receptor of bacterial chemotaxis and the trp repressor of E. coli. Implications for transmembrane signaling.
A high resolution structure of the N-terminal ligand-binding domain of the aspartate receptor which mediates aspartate chemotaxis in Salmonella typhimurium has recently been reported. A least-squares superposition of the alpha-amino nitrogen, alpha-carbon, beta-carbon, and alpha-carboxylate carbon of the aspartate bound to the aspartate receptor onto the equivalent atoms in the tryptophan bound to the trp repressor provides evidence for similarity between key parts of the active sites that bind to the alpha-amino and alpha-carboxylates of the respective ligands. Because the N-terminal domain of the aspartate receptor and the trp repressor also share other structural similarities, we hypothesize that the similarity between the aspartate receptor and the trp repressor derives from a similarity in ligand-induced conformational changes at the active sites of these proteins. This hypothesis also implies that an important signaling event in the aspartate receptor occurs through tertiary conformational changes within a single subunit.
Aspartic Acid↗
Economic impact report.
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Animal Husbandry↗
DNA fingerprinting and eyewitness testimony.
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DNA Fingerprinting↗
The biochemistry of the neuron: methods and calculations for the analysis of neurotransmitter secretion from populations of single cells.
Methods and calculations for the continuous measurement of secretion of radiolabeled neurotransmitter from cultured neuronal cells are demonstrated. The method is used to measure the secretion of [3H]-norepinephrine by neuronally differentiated PC12 cells in response to a stepwise presentation of a depolarizing stimulus. The response is known to be biphasic, consisting of a transient burst of secretion (phase I) followed by a plateau of secretion (phase II). Habituation, in which cellular secretion is lowered by repetitive stimulation of the cells, is shown here to lower uniformly both phases of secretion. There thus appears to be a mechanism within the cell that holds constant the proportions of phase I and phase II secretion even though the overall size of the secretory response may be regulated as a consequence of repetitive stimulation. The feasibility of the method for widespread application to cultured neuronal cells is demonstrated.
Animals↗
Elephants, monstrosities, and the law.
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Humans↗
Receptors and transmembrane signaling.
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Animals↗
Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription.
The NtrC transcription factor is a member of a family of homologous prokaryotic regulatory proteins that participate in the transduction of extracellular and nutritional signals. It has been demonstrated that the phosphate group from a histidine residue of the phosphorylated NtrB protein autokinase is transferred to the NtrC protein. Phosphorylation of the NtrC protein is transient and activates its transcriptional enhancement activity. We have investigated the site of phosphorylation of the Salmonella typhimurium NtrC protein and find that it is an aspartate residue (Asp-54) that is found within a sequence conserved in all of the members of the family of regulatory proteins. We propose that this phosphorylation is an NtrC protein histidine phosphatase catalytic intermediate. This conclusion suggests that the NtrC family should be viewed not as kinase substrates but as enzymes that can catalyze the hydrolysis of their activated forms in a concentration-independent fashion. They are similar in this sense to eukaryotic signal-transducing GTPases.
Adenosine Triphosphatases↗
Effect of an induced conformational change on the physical properties of two chemotactic receptor molecules.
The physical properties and conformational dynamics of the Salmonella typhimurium ribose and galactose receptors have been examined. Studies involving circular dichroism, fluorescence, absorption spectroscopy, and sedimentation analysis show that the two receptor proteins have different morphologies and exhibit diverse responses to sugar binding. The ribose receptor lacks both tryptophan and disulfide residues, and the galactose receptor lacks disulfides and has only a single tryptophan residue. By virtue of these fortuitous properties, the conformational changes induced in these proteins by sugar binding can be dissected by utilizing a variety of physical probes. A ligand-induced conformational change in the ribose receptor is shown by circular dichroism and fluorescence spectroscopy, which reveal spectral changes assignable to tyrosine, phenylalanine, and methionine residues. A conformational change in the galactose receptor has been demonstrated by fluorescence spectroscopy involving the distant reporter group method, which shows changes assignable to tryptophan and methionine sites and which is corroborated by sedimentation analysis. It is clear that there are extensive conformational changes in the two receptor proteins and that the different physical methods provide complementary information on the nature of these changes.
Amino Acids↗
Membrane receptors for aspartate and serine in bacterial chemotaxis.
High affinity binding sites for serine and aspartate have been characterized in membranes from Salmonella typhimurium and Escherichia coli. Greater than 80% of these sites have been identified as chemotaxis receptors. Mutants lacking binding sites for these amino acids have been shown to have corresponding defects in taxis. The substrate specificity of each of the receptors in Salmonella is very high; most analogs of serine and aspartate do not bind to these receptor sites and do not affect chemotaxis. The transport of these amino acids is apparently not related to chemotaxis. At least 2500 serine receptors and 1200 aspartate receptors with dissociation constants of about 5 microM are present in the membrane fraction of logarithmically growing cells.
Aspartic Acid↗