Hairpin-shaped DNA duplexes with disulfide bonds in sugar-phosphate backbone as potential DNA reagents for crosslinking with proteins. (1/267)

Convenient approaches were described to incorporate -OP(=O)O(-)-SS-O(-)(O=)PO- bridges in hairpin-shaped DNA duplexes instead of regular phosphodiester linkages: (i) H2O2- or 2,2'-dipyridyldisulfide-mediated coupling of 3'- and 5'-thiophosphorylated oligonucleotides on complementary template and (ii) more selective template-guided autoligation of a preactivated oligonucleotide derivative with an oligomer carrying a terminal thiophosphoryl group. Dithiothreitol was found to cleave completely modified internucleotide linkage releasing starting oligonucleotides. The presence of complementary template as an intrinsic element of the molecule protects the hairpin DNA analog from spontaneous exchange of disulfide-linked oligomer fragments and makes it a good candidate for auto-crosslinking with cysteine-containing proteins.  (+info)

The effect of carboxyl group modification on the chromophore regeneration of archaeopsin-1 and bacterioopsin. (2/267)

Carboxyl group modification with DCCD and NCD-4 was employed to investigate the chemical environment of the side chains of archaeopsin-1 (aO-1) and bacterioopsin (bO). Some differences were observed between aO-1 and bO. Although DCCD or NCD-4 did not modify aO-1 in bleached membrane, they modified bO in bleached membrane and in mixed DMPC/CHAPS/SDS micelles at neutral pH, thereby affecting the opsin shift and the photocycle of the regenerated chromophore. On the contrary, after solubilization with SDS, aO-1 and bO were modified by DCCD and NCD-4, which decreased the chromophore regeneration. In particular, the reaction of aO-1 in SDS with NCD-4 proceeded in a 1:1 ratio at neutral pH. The fluorescence and CD spectra indicated that the modified site was located in the hydrophobic, asymmetrical region. Lysyl-endopeptidase digestion of NCD-4 modified aO-1 produced a fluorescent fragment and amino acid sequence analysis showed that Asp85 or Asp96 in helix C is a probable candidate for the modified residue at present. Kinetic CD measurements revealed that the introduction of N-acylurea at an Asp residue in helix C did not affect the formation of the transient intermediate but inhibited the side chain packing during refolding.  (+info)

Active site characterization of RNase Rs from Rhizopus stolonifer: involvement of histidine and lysine in catalysis and carboxylate in substrate binding. (3/267)

Chemical modification studies on purified RNase Rs revealed the involvement of a single histidine, lysine and carboxylate residue in the catalytic activity of the enzyme. RNA could not protect the enzyme against DEP- and TNBS-mediated inactivation whereas, substrate protection was observed in case of EDAC-mediated inactivation of the enzyme. K(m) and k(cat) values of the partially inactivated enzyme samples suggested that while histidine and lysine are involved in catalysis, carboxylate is involved in substrate binding. Active site nature of RNase Rs suggests that the inability of the enzyme to readily convert 2',3'-cyclic nucleotides to 3'-mononucleotides is probably due to the absence of catalytically active second histidine residue.  (+info)

Structural and kinetic studies of the pyruvate-ferredoxin oxidoreductase/ferredoxin complex from Desulfovibrio africanus. (4/267)

The pyruvate-ferredoxin oxidoreductase (PFOR)/ferredoxin (Fd) system of Desulfovibrio africanus has been investigated with the aim of understanding more fully protein-protein interaction and the kinetic characteristics of electron transfer between the two redox partners. D. africanus contains three Fds (Fd I, Fd II and Fd III) able to function as electron acceptors for PFOR. The complete amino acid sequence of Fd II was determined by automatic Edman degradation. It revealed a striking similarity to that of Fd I. The protein consists of 64 residues and its amino acid sequence is in agreement with a molecular mass of 6822.5 Da as measured by electrospray MS. Fd II contains five cysteine residues of which the first four (Cys11, Cys14, Cys17 and Cys54) are likely ligands for the single [4Fe-4S] cluster. A covalently cross-linked complex between PFOR and Fd I or Fd II was obtained by using a water soluble carbodiimide. This complex exhibited a stoichiometry of one ferredoxin for one PFOR subunit and is dependent on the ionic strength. The second-order rate constants for electron transfer between PFOR and Fds determined electrochemically using cyclic voltammetry are 7 x 107 M-1.s-1 for Fd I and 2 x 107 M-1.s-1 for Fd II and Fd III. The Km values of PFOR for Fd I and Fd II measured both by the electrochemical and the spectrophotometric method have been found to be 3 microM and 5 microM, respectively. The three-dimensional modelling of Fd II and surface analysis of Fd I, Fd II and PFOR suggest that a protein-protein complex is likely to be formed between aspartic acid/glutamic acid invariant residues of Fds and lysine residues surrounding the distal [4Fe-4S] cluster of PFOR. All of these studies are indicative of the involvement of electrostatic interactions between the two redox partners.  (+info)

S-layer-coated liposomes as a versatile system for entrapping and binding target molecules. (5/267)

In the present study, unilamellar liposomes coated with the crystalline bacterial cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were used as matrix for defined binding of functional molecules via the avidin- or streptavidin-biotin bridge. The liposomes were composed of dipalmitoyl phosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4 and they had an average size of 180 nm. For introducing specific functions into the S-layer lattice without affecting substances encapsulated within the liposomes, crosslinking and activation reagents had to be identified which did not penetrate the liposomal membrane. Among different reagents, a hydrophilic dialdehyde generated by periodate cleavage of raffinose and a sulfo-succinimide activated dicarboxylic acid were found to be impermeable for the liposomal membrane. Both reagents completely crosslinked the S-layer lattice without interfering with its regular structure. Biotinylation of S-layer-coated liposomes was achieved by coupling p-diazobenzoyl biocytin which preferably reacts with the phenolic residue of tyrosine or with the imidazole ring of histidine. By applying this method, two biotin residues accessible for subsequent avidin binding were introduced per S-layer subunit. As visualized by labeling with biotinylated ferritin, an ordered monomolecular layer of streptavidin was formed on the surface of the S-layer-coated liposomes. As a second model system, biotinylated anti-human IgG was attached via the streptavidin bridge to the biotinylated S-layer-coated liposomes. The biological activity of the bound anti-human IgG was confirmed by ELISA.  (+info)

An essential glutamyl residue in EmrE, a multidrug antiporter from Escherichia coli. (6/267)

EmrE is an Escherichia coli 12-kDa protein that confers resistance to toxic compounds, by actively removing them in exchange with protons. The protein includes eight charged residues. Seven of these residues are located in the hydrophilic loops and can be replaced with either Cys or another amino acid bearing the same charge, without impairing transport activity. Glu-14 is the only charged residue in the membrane domain and is conserved in all the proteins of the family. We show here that this residue is the site of action of dicyclohexylcarbodiimide, a carbodiimide known to act in hydrophobic environments. When Glu-14 was replaced with either Cys or Asp, resistance was abolished. Whereas the E14C mutant displays no transport activity, the E14D protein shows efflux and exchange at rates about 30-50% that of the wild type. The maximal DeltapH-driven uptake rate of E14D is only 10% that of the wild type. The mutant shows a different pH profile in all the transport modes. Our results support the notion that Glu-14 is an essential part of a binding domain shared by substrates and protons but mutually exclusive in time. This notion provides the molecular basis for the obligatory exchange catalyzed by EmrE.  (+info)

Identification and characterization of a novel cAMP receptor protein in the cyanobacterium Synechocystis sp. PCC 6803. (7/267)

Three open reading frames of Synechocystis sp. PCC 6803 encoding a domain homologous with the cAMP binding domain of bacterial cAMP receptor protein were analyzed. These three open reading frames, sll1371, sll1924, and slr0593, which were named sycrp1, sycrp2, and sypk, respectively, were expressed in Escherichia coli as His-tagged or glutathione S-transferase fusion proteins and purified, and their biochemical properties were investigated. The results obtained for equilibrium dialysis measurements using these recombinant proteins suggest that SYCRP1 and SYPK show a binding affinity for cAMP while SYCRP2 does not. The dissociation constant of His-tagged SYCRP1 for cAMP is approximately 3 microM. A cross-linking experiment using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide revealed that His-tagged SYCRP1 forms a homodimer, and the presence or absence of cAMP does not affect the formation of the homodimer. The amino acid sequence reveals that SYCRP1 has a domain similar to the DNA binding domain of bacterial cAMP receptor protein in the COOH-terminal region. Consistent with this, His-tagged SYCRP1 forms a complex with DNA that contains the consensus sequence for E. coli cAMP receptor protein in the presence of cAMP. These results strongly suggest that SYCRP1 is a novel cAMP receptor protein.  (+info)

Comparison of the efficiency of various coupling systems in the acylation of model secondary amines with thymin-1-ylacetic acid. (8/267)

Peptide nucleic acids (PNAs) make a promising group of DNA analogues. The backbone of typical PNA oligomers is composed of N-(2-aminoethyl)glycine units, linked by the peptide bonds. The backbone secondary amine groups are acylated with carboxyalkyl derivatives of nucleobases. One of the PNA synthesis step causing some problems is the acylation of the monomer backbone with the nucleobase derivatives. The aim of the study was to compare the efficiency of various coupling systems in the acylation. Simple model compounds (piperidine and proline) were used, as well as equimolar amounts of the coupling reagents. Selected systems based on carbodiimides, aminium or phosphonium salts, mixed anhydride, and active esters were tested.  (+info)

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