Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry.
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Integration of biochemical and biophysical data on the lactose permease of Escherichia coli has culminated in a molecular model that predicts substrate-protein proximities which include interaction of a hydroxyl group in the galactopyranosyl ring with Glu269. In order to test this hypothesis, we studied covalent modification of carboxyl groups with carbodiimides using electrospray ionization mass spectrometry (ESI-MS) and demonstrate that substrate protects the permease against carbodiimide reactivity. Further more, a significant proportion of the decrease in carbodiimide reactivity occurs specifically in a nanopeptide containing Glu269. In contrast, carbodiimide reactivity of mutant Glu269-->Asp that exhibits lower affinity is unaffected by substrate. By monitoring the ability of different substrate analogs to protect against carbodiimide modification of Glu269, it is suggested that the C-3 OH group of the galactopyranosyl ring may play an important role in specificity, possibly by H-bonding with Glu269. The approach demonstrates that mass spectrometry can provide a powerful means of analyzing ligand interactions with integral membrane proteins. (+info)
Identification of the dicyclohexylcarbodiimide-reactive protein component of the adenosine 5'-triphosphate energy-transducing system of Escherichia coli.
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Membranes of Escherichia coli contain an adenosine 5'-triphosphate (ATP) energy-transducing system that is inhibited by treatment with dicyclohexylcarbodiimide (DCCD). The carbodiimide-reactive protein component of this system has been identified after treatment with [14C]DCCD. This protein has an apparent molecular weight of 9,000 as judged from acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is extracted from the membrane with chloroform-methanol (2:1). These properties are similar to the analogous protein previously identified in mitochondria (Cattell et al., 1971). A mutant strain, RF-7, has been isolated which derives energy from oxidative phosphorylation in the presence of 5 mM DCCD. The ATP hydrolase activity of the membraned system in the mutant was considerably less sensitive to inhibition by DCCD than that in the wild type. The carbodiimide-reactive protein, which was easily labeled by [14C]DCCD in the wild type, was labeled much less rapidly in the carbodiimide-resistant mutant. It is thus concluded that the reaction of DCCD with this specific protein leads to inhibition of the ATP energy-transducing reactions. The mutation causing carbodiimide resistance in strain RF-7 was mapped. It is cotransduced with the uncA gene at a frequency exceeding 90%. The mutationally altered protein causing the carbodiimide resistance was not conclusively identified. However, reconstitution experiments indicate that the altered protein is not one of the subunits of the soluble ATP hydrolase activity, which can be removed from the membrane by washing with 1 mM tris(hydroxymethyl)aminomethane buffer lacking Mg2+. The carbodiimide-reactive protein remains with the membrane residue after removal of the soluble ATP hydrolase and is thus distinct from these subunits as well. (+info)
Condensation reaction of hexanucleotides containing guanine and cytosine with water soluble carbodiimide.
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The condensation reactions of hexanucleotides involving guanine and cytosine in the presence of water-soluble carbodiimide (WSC) have been investigated as a model reaction of the prebiotic formation of RNA under primitive earth. The reactions formed cyclic hexanucleotides and dimers in which the product yields were dependent on the sequence. (+info)
Role of the M-loop and reactive center loop domains in the folding and bridging of nucleosome arrays by MENT.
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MENT is a developmentally regulated heterochromatin-associated protein that condenses chromatin in terminally differentiated avian blood cells. Its homology to the serpin protein family suggests that the conserved serpin reactive center loop (RCL) and the unique M-loop are important for its function. To examine the role of these domains, we studied the interaction of wild-type and mutant MENT with naked DNA and biochemically defined nucleosome arrays reconstituted from 12-mer repeats containing nucleosome positioning sequences. Wild-type MENT folded the naked DNA duplexes into closely juxtaposed parallel structures ("tramlines"). Deletion of the M-loop, but not inactivation of the RCL, prevented tramline formation and the cooperative interaction of MENT with DNA. Reconstitution of wild-type MENT with nucleosome arrays caused their tight folding and self-association. M-loop deletion inhibited nucleosome array folding, whereas the inactive RCL mutant was competent to fold the nucleosome arrays, but had a significantly impaired ability to cause their self-association. Bifunctional chemical cross-linking of MENT revealed oligomerization of wild-type MENT in the presence of chromatin and DNA. This oligomerization was severely reduced in the RCL mutant. We propose that the mechanism of MENT-induced heterochromatin formation involves two independent events: bringing together nucleosome linkers within a chromatin fiber and formation of protein bridges between chromatin fibers. Ordered binding of MENT to linker DNA via its unique M-loop domain promotes the folding of chromatin, whereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL. (+info)
Carboxylic acid-modified polyethylene: a novel support for the covalent immobilization of polypeptides for C-terminal sequencing.
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We have developed a method for the covalent immobilization of peptides, for the purpose of C-terminal sequencing, to a novel solid support, carboxylic acid-modified polyethylene (PE-COOH) film. The peptides are attached by coupling the N-terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3-diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly thorough the alpha-amino group for the peptide, SIGSLAK, which after attachment to the PE-COOH support permitted the C-terminal lysine residue to be sequenced in good yield, indicating that the epsilon-amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C-terminal sequencing including (1) stability to base and the high temperatures (65 degrees C) employed for C-terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 x 5-mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N-terminal sequencing (Shively, J.E., Miller, P., & Ronk, M., 1987, Anal. Biochem. 163, 517-529). Automated C-terminal sequencing on these supports is described in the companion paper (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). (+info)
Retinoic acid regulates both expression of the nerve growth factor receptor and sensitivity to nerve growth factor.
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In PC12 cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component of the nerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of 125I-labeled NGF to p75NGFR, and an increase in the binding of NGF to low affinity sites. RA-treated cells are more sensitive to NGF, but not to either fibroblast growth factor or phorbol 12-myristate 13-acetate, showing that RA has a specific effect on the responsiveness of PC12 cells to NGF. Exposure to RA leads neither to an increase in the expression of mRNA for trk, another component of the NGF receptor, nor to an increase in binding to high affinity receptors, suggesting that an increase in the expression of p75NGFR is sufficient to make cells more sensitive to NGF. This work suggests that, in addition to having direct effects on gene expression, RA can indirectly modulate differentiation of neurons by modifying their expression of cell surface receptors to peptide growth factors. (+info)
Partial purification of a dicyclohexylcarbodi-imide-sensitive membrane adenosine triphosphatase complex from the obligately anaerobic bacterium Clostridium Pasteurianum.
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The membrane adenosine triphosphatase complex of vegetatively growing Clostridium pasteurianum, solublized with Triton X-100, has been recovered as a significantly purified particulate preparation that is still sensitive to inhibition by dicyclohexylcarbodiimide and butyricin 7423. (+info)
Localization and possible role of an adenosine triphosphatase in Chlorobium thiosulfatophilum.
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1. Evidence is presented that the ATPase activity detected in cell extracts of Chlorobium thiosulfatophilum is bound to the cytoplasmic membrane rather than to the chlorobium vesicles. 2. The activity of this ATPase is inhibited in vitro by various carbodiimides, phloridzin and sodium azide. 3. The apparent Km for ATP is approximately 0.2 mM and the enzyme shows product inhibition by ADP. 4. Photophosphorylation, characterized in vivo, is inhibited by many of the compounds that inhibit the ATPase. (+info)