ADP Stabilizes the Human Rad51-single stranded DNA Complex and Promotes Its DNA Annealing Activity

Background: Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single-stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins. Results: Measurements using the fluorescence ssDNA analogue, poly(1,N (6) -etheno-deoxyadenosine), indicate that ATP affects the HsRad51-ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state. While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51-ssDNA complex. ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs. Conclusions: The hydrolysis of ATP promotes a transition of the HsRad51-ssDNA complex from a stiff state to less stiff state. The first state may be important for the strand separation of dsDNA in the initial step of strand exchange, while the second state may be important for annealing in the next step. However, hydrolysis does not dissociate HsRad51 from DNA as a component step of its recycling

Morphology and molecular conformation in thin films of poly-gamma-methyl-L-glutamate at the air-water interface

The behavior of poly-gamma-methyl-L-glutamate (pMeE) at the air-water interface has been studied with the surface film balance technique. In addition, Langmuir-Blodgett (LB) films of pMeE deposited on mica and quartz have been studied by atomic force microscopy (AFM) and circular and linear dichroism (CD and LD) spectroscopy. Depending on the spreading solvent, pMeE displays strikingly different compression isotherms. When spread from chloroform or trifluoroacetic acid (TFA) the surface pressure isotherms are consistent with that of a peptide in a-helix conformation. However, the latter solvent gives rise to isotherms with a considerably smaller apparent mean molecular area, A(0). When spread from pyridine, on the other hand, pMeE yields an isotherm that is expanded and inconsistent with the presence of a monolayer consisting entirely of a-helical peptides. Isotherms and AFM images strongly suggest that peptide aggregation and solvent retention are the main factors behind the isotherm differences. When the water-soluble spreading solvent TFA is used, pMeE forms discrete wormlike aggregates embedded in a monolayer matrix. In the pyridine case, aggregation in the spreading solvent and retention of pyridine in the film result in a rough aggregate network coexisting with discrete aggregates. No aggregation takes place when chloroform is used as spreading solvent. CD and LD spectra of the LB films reveal a pronounced lateral orientation of the alpha-helices in films spread from chloroform and TFA, while spectra of films spread from pyridine are consistent with unordered peptide strands in beta-sheet conformation. In conclusion, the results show that if water-soluble and/or low-volatile solvents are used as spreading media, hydrophobic peptides cannot, a priori, be assumed to form proper monolayers

Synthesis and fluorescence properties of novel transmembrane probes and determination of their orientation within vesicles

Two novel transmembrane fluorescent diester probes D and E bearing an anthracenediyl moiety in the middle of the molecule have been synthesized. Their absorption and fluorescence spectra in CHCl3 solution as well as their fluorescence characteristics in dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles were determined. Although their absorption spectra (first transition, S-0 --> S-1) present a good overlap with the fluorescence spectrum of tryptophan, only probe E could be a good acceptor for the energy-transfer experiments, since a strong overlap exists between the absorption spectrum of tryptophan and the second transition (S-0 --> S-2) of the absorption spectrum of probe D. The Forster critical distance R-0 for energy transfer between tryptophan (donor) and probe E (acceptor) is found to be 23-24 Angstrom. Finally, linear-dichroism studies on shear-deformed DMPC vesicles show the incorporated probe E to lie essentially perpendicular to the bilayer plane. These results establish that probe E could be useful in the study of membrane-bound protein topography by the fluorescence-energy-transfer method