Binding specificity of mutant V55C disulfide bonded dimeric λ-Cro protein (CroVC) to double-stranded DNA (dsDNA) was studied using generic hexamer oligonucleotide microchip. The curves of dissociation of hybridized DNA in the presence and absence of CroVC were converted into the effective discriminant constants to assess the relevant thermodynamic equilibrium binding constants for dsDNA-protein complexes. Then, tiling of longer oligonucleotides with shorter oligomers was used to search for sequence motifs with the highest binding specificity similarly to sequencing by hybridization. The comparison of the deduced sequences with the known natural operator half-sites demonstrated the principal ability to discern and reconstruct the major parts of 7-mer motifs corresponding to the strongest binding of CroVC subunits. Our results show the applicability of generic microchips to the analysis of binding specificity in the case of multi-subunit DNA-binding proteins.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biomolecular Structure and Dynamics|
|State||Published - Dec 2003|
Bibliographical noteFunding Information:
The authors are very thankful to A. S. Zasedatelev and to A. V. Finkelstein and members of his seminar at the Institute of Protein Research for useful discussions and valuable remarks. We are also grateful to G. M. Ershov for the chip manufacturing, to D. Zamyatkin, Yu. Kolyagin, and members of protein thermodynamics group of the Institute of Protein Research for their help in CroVC purification. Our special thanks are to A. Kolchinsky from Health Front Line, Ltd. (Champaign, IL) for his excellent editorial assistance. This work was supported by grant 02-04-48814 of Russian Foundation for Fundamental Research.