Oligomerization status of the TmaSSB and

TneSSB proteins Analysis of the purified proteins by SDS-PAGE revealed a single major band with a molecular mass of about 16 kDa for both proteins. In contrast, analysis by gel filtration chromatography revealed single peaks with a molecular mass of about Selleckchem Small molecule library 60.48 kDa for TmaSSB and 61.86 kDa for TneSSB (Figure 3). This native molecular mass is approximately is 3.7 times the molecular mass of the monomer for both proteins. This confirmed our prediction that in solution the TmaSSB and TneSSB proteins exist as homotetramers. Chemical cross-linking using glutaraldehyde confirmed the tetrameric state of the examined proteins (not shown). Figure 3 Analytical gel filtration of Tma SSB and Tne SSB on Superdex HR 75 column. A standard linear regression curve was generated by plotting the log of

the molecular mass of the calibration proteins against their retention times (min) and is shown. The calibration proteins include bovine albumin (66 kDa), ovalbumin (43 kDa), carbon anhydrase (29 kDa) and cytochrome C (12.4 kDa). DNA-binding properties When (dT)35, (dT)60 or (dT)76 were incubated with increasing amounts of TmaSSB or TneSSB, a single band of reduced mobility was observed (Figure 4, complex I). Most of those oligonucleotides were shifted after addition

of 10 pmol of SSBs, and the Akt inhibitor mobility of the shifted band remained constant at the higher protein amounts (100 pmol). One band of identical mobility was observed for (dT)120 at the low protein amounts, but a second band with a lower mobility appeared at the higher protein amounts (100 pmol; PTK6 Figure 4, complex II)). These results suggest that TmaSSB and TneSSB bind to (dT)35, (dT)60 or (dT)76 as one single homotetramer whereas two SSB homotetramers bind to (dT)120. Similar binding patterns were observed with the TmaSSB and TneSSB proteins in different salt concentrations (2 or 100 mM NaCl). Figure 4 Binding of Tma SSB and Tne SSB to oligo(dT) and to M13 ssDNA- gel mobility shift assays. The binding of the TmaSSB and TneSSB proteins to the naturally occurring circular M13 ssDNA (6,407 nucleotides) was also examined. In this experiment, a fixed amount of M13 ssDNA was incubated with increasing amounts of SSB protein, and the resulting complexes were analyzed by agarose gel electrophoresis (Figure 4). When increasing amounts of TmaSSB or TneSSB protein were added to M13 ssDNA, there was a progressive decrease in the mobility of the M13 ssDNA. To further explore the binding properties of the examined SSB proteins, we used fluorescence spectroscopy.