VirE2-dependent pores for ssDNA transfer across artificial and cell membranes.
The transfer of single-stranded (ss) T-DNA from soil bacteria of the genus Agrobacterium with the help of the VirE2 protein, which possibly mediates the delivery of ss-T-DNA across the cell membrane, was demonstrated earlier, but how VirE2 participates in ssDNA transfer across artificial and natural membranes is not known. Using computational methods, we reconstructed model structures composed of two and four VirE2 proteins and showed by the MOLE program the formation of pores with channel diameters of 1.2-1.6 and 1.4-4.6 nm in a model structure formed from two and four VirE2 molecules, respectively. Using light scattering, we recorded the size distribution for recombinant VirE2-dependent complexes in aqueous solutions and found that VirE2 in a buffer solution is present as a complex made up of two or more proteins. We revealed single, long-lived jumps in voltage-dependent membrane conductance during coincubation of planar black membranes with the VirE2 protein. On the addition of VirE2 and FAM-labeled oligonucleotides to HeLa cells, the fluorescence intensity for the cells increased by 56% as compared to that for cells incubated only with oligonucleotides.
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia.
Irina
I
Volokhina
Yury
Y
Gusev
Svyatoslav
S
Mazilov
Mikhail
M
Chumakov
D060054:Agrobacterium / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007473:Ion Channels; C036483:T-DNA; C051583:virE2 protein, Agrobacterium