Complexation and phase transfer of nucleotides by gramicidin S

Gramicidin S (GrS), an amphiphilic cyclosymmetric decapeptide produced by Bacillus brevis G-B and Nagano, binds nucleotides in water to yield a complex which partitions into organic solvents. The observed phase-transfer efficiencies at a given pH increase in the order AMP less than ADP less than ATP. The lipophilic complexes have well-defined stoichiometries, which were determined to be 1:1 for ADP-GrS at pH 7 and ATP-GrS at pH 3 and 1:2 for ATP-GrS at pH 7. The interaction is primarily ionic, involving coordination of the ornithine N delta H3+ groups of the peptide and the phosphoryl groups of the nucleotide, with little contribution from the nucleoside moiety. Exchange of organic and inorganic phosphates was also found to be mediated by GrS. The nucleotide complexes are sparingly soluble in water and self-associate extensively in CHCl3, most likely by cross-beta-aggregation, to yield large, ribbonlike aggregates which give rise to broad NMR resonances. Structures for the 1:1 and 1:2 complexes are proposed. In the latter, two GrS molecules envelop the nucleotide, orienting their apolar faces externally in opposite directions, while the lateral faces retain considerable polar character and direct aggregation in organic media. The 1:1 complex possesses a single apolar face and is less lipophilic. Binding constants were estimated by simulation of the extraction data. For the 1:1 complexes, K1:1 congruent to 4 X 10(4) M-1 for either ADP or ATP. Phase transfer of the ATP complex at pH 7 could be modeled either by stochastically independent binding to two noninteracting sites on the nucleotide with K1 approximately K2 approximately K1:1 or by a sequential process with K1 approximately K1:1 and K2/K1 less than 100. It is concluded that the apparent selectivity of GrS for ATP over ADP is a consequence of the greater lipophilicity and tendency to aggregate of the 1:2 complex, rather than an intrinsically higher binding affinity for triphosphates. GrS is, to our knowledge, the first peptide known to possess phase-transfer activity toward nucleotides; this is, in addition, the first molecular recognition process in which GrS is demonstrated to participate in vitro at physiologically active concentrations.     

Biosynthesis of gramicidin S with the aid of dipeptides by gramicidin S synthetase.

Dipeptides L-phenylalanyl-proline, D-phenylalanyl-proline, prolyl-valine, valyl-lysine, lysyl-leucine and leucyl-phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p-nitrophenyl esters of D-phenylalanyl-proline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone.     

Liposome-mediated transfer of nucleic acids in plant protoplasts

Liposomes spontaneously interact with the plasma membrane of protoplasts. The delivery of their content to the protoplast, however, has to be induced by physical or chemical treatments. Three major approaches have been used to study and optimize the delivery of RNA or DNA molecules: viral infection, transient gene expression and stable transformation. Conclusive evidence for the delivery of nuclei acids to the treated protoplasts has been obtained. Stable transformants expressing kanamycin resistance have been further characterized by hybridization techniques and progeney analysis. The potential and limitations of this approach for the transformation of higher plants is discussed.     

Information transfer from DNA to peptide nucleic acids by template-directed syntheses.

Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.     

Information transfer from peptide nucleic acids to RNA by template-directed syntheses.

Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.     

Mode of antibacterial action by gramicidin S

To elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experiment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 microgram/ml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6 micrograms/ml, and the cells were temporarily saturated with GS at 10 micrograms/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by adsorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. coli increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall.     

Control of electron transfer in DNA by peptide nucleic acids (PNA).

The one-electron oxidation of PNA-DNA hybrid containing G-triplet sequence was examined. In DNA duplex G-triplet was selectively cleaved by oxidation, whereas in PNA-DNA hybrid cleavage efficiency was extremely lowered. These result suggested that cleavage efficiency of PNA-DNA hybrid was different from that of B-form DNA duplex.     

Conformation of gramicidin S

A molecular conformation of Gramicidin S was derived on the basis of conformational calculations taking into account the available experimental data. The conformation is characterized by a dyad axis which relates the two chemically equivalent halves of the molecule and contains four hydrogen bonds; other structural features agree with experimental results. X Ray Crystallographic evidences for the relative position of the Ornithine residues is also reported which supports an important feature of the structure of Gramicidin S.     

Fluorescence resonance energy transfer in the study of nucleic acids

Recent data are reviewed on the employment of fluorescence resonance energy transfer (FRET) in studying hybridization and higher structures of nucleic acids as well as their enzyme- and ribozyme-catalyzed reactions.     

Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.     

Fluorescence resonance energy transfer as a structural tool for nucleic acids

Fluorescence resonance energy transfer is a spectroscopic method that provides distance information on macromolecules in solution in the range 20-80 A. It is particularly suited to the analysis of the global structure of nucleic acids because the long-range distance information provides constraints when modelling these important structures. The application of fluorescence resonance energy transfer to nucleic acid structure has seen a resurgence of interest in the past decade, which continues to increase. An especially exciting development is the recent extension to single-molecule studies.     

Hydrolysis of nucleic acids by sepharose-micrococcal endonuclease

Initial reaction rates for the hydrolysis of nucleic acids with micrococcal endonuclease (EC 3.1.31.1) insolubilized on Sepharose are strongly influenced by diffusional limitations. Although the absolute values are low, they can be increased substantially by changing particle and pore size of the support, or enzyme concentration in the insoluble derivative. As a result of steric and diffusional limitations, the course of the reaction and selectivity to hydrolysis products for the insoluble derivatives are different to those of the native enzyme; the former produces mainly large and small fragments but few of intermediate size. Because of these differences in course and selectivity of the reaction, diffusional limitations become less important when high initial reaction rates are not required.