Single stranded binding protein: Difference between revisions

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Residues involved in ssDNA binding
Residues involved in ssDNA binding
The Trp fluorescence of SSB is quenched by approx90% upon binding poly(dT) in the (SSB)65 binding mode5, 6. However, in the (SSB)35 mode, Trp fluorescence is quenched by only approx50% due to the fact that only half of the Trp residues interact with DNA in this binding mode11 (Fig. 4b). Spectroscopic studies also suggest that Trp 40 and Trp 54 form stacking interactions with the bases13, 14. Mutagenesis of Trp 40 and Trp 54 reduce ssDNA binding affinity14, 15. Crosslinking experiments16 and mutational studies17, 18 have shown that Phe 60 is involved in DNA binding. Consistent with these observations, Trp 40, Trp 54 and Phe 60, in the structure of the SSBc−ssDNA complex, make extensive interactions with the ssDNA (see above).
Spectroscopic studies also suggest that Trp 40 and Trp 54 form stacking interactions with the bases13, 14. Mutagenesis of Trp 40 and Trp 54 reduce ssDNA binding affinity14, 15. Crosslinking experiments16 and mutational studies17, 18 have shown that Phe 60 is involved in DNA binding. Consistent with these observations, Trp 40, Trp 54 and Phe 60, in the structure of the SSBc−ssDNA complex, make extensive interactions with the ssDNA (see above).


Only a few natural ssb mutants have been isolated and characterized; two of them, ssb-1 and ssb-3, are located within SSBc. The ssb-1 mutant, a H55Y mutation, destabilizes the SSB tetramer to favor monomers1, 2, 12. The ssb-3 mutant, a G15D mutation, renders the cell extremely sensitive to UV19. In the model of the SSBc−ssDNA structure presented here, Gly 15 is within 3.5 Å of the phosphate backbone at C4 (Fig. 2a). Mutation to Asp may sterically hinder ssDNA binding. The structure therefore suggests that G15D would affect ssDNA binding.
In the model of the SSBc−ssDNA structure presented here, Gly 15 is within 3.5 Å of the phosphate backbone at C4 (Fig. 2a). Mutation to Asp may sterically hinder ssDNA binding. The structure therefore suggests that G15D would affect ssDNA binding.


Finally, thermodynamic studies indicate that electrostatic interactions have a major role in SSB−ssDNA binding11, 20. The role of Lys residues and the N-terminus in ssDNA binding has also been probed by chemical modification21 and it was observed that acetylation of Lys 43, Lys 62, Lys 73, Lys 87, and the terminal amine is greatly reduced upon binding ssDNA. In the structure, these Lys residues, as well as the N-terminal amine, are within contact distance of the ssDNA backbone and selective acetylation of these residues would be expected to have a significant effect on ssDNA binding. Other basic residues make interactions with the ssDNA, either with the ssDNA bases (Arg 3) or with the phosphate backbone (Arg 84).
Finally, thermodynamic studies indicate that electrostatic interactions have a major role in SSB−ssDNA binding11, 20. The role of Lys residues and the N-terminus in ssDNA binding has also been probed by chemical modification21 and it was observed that acetylation of Lys 43, Lys 62, Lys 73, Lys 87, and the terminal amine is greatly reduced upon binding ssDNA. In the structure, these Lys residues, as well as the N-terminal amine, are within contact distance of the ssDNA backbone and selective acetylation of these residues would be expected to have a significant effect on ssDNA binding. Other basic residues make interactions with the ssDNA, either with the ssDNA bases (Arg 3) or with the phosphate backbone (Arg 84).
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