ИСТИНА

The conjugation of proteins with oligodeoxyribonucleotides often considerably changes their properties. Fusion with oligonucleotides can extend the specificity of restriction endonucleases, which is a necessary property for targeting individual genes in complex genomes [Eisenschmidt K. et al. Nucleic Acids Res. 2005, 33, 7039]. The aim of this work was to construct an enzyme with pronounced thermo-dependent properties that could later serve as a model to understand the mechanism of naturally occurring thermo-dependent processes. We chose a well-studied homodimeric type II restriction endonuclease SsoII (R.SsoII) as a model system. We developed the “molecular gate” strategy based on the fact that most type II restriction endonucleases are homodimers [Hien L.T. et al. Bioconjug. Chem. 2011, 22, 1366]. The DNA-binding center is located in the interface between the two subunits. It is possible to modify the protein at the entrance of the DNA-binding site and block its activity. To create the obstacle for DNA penetration to the active center we suggested to attach oligonucleotides to the surface of the enzyme and use their thermo-dependent ability to form a duplex. The duplex is suggested to be stable at room temperature, thus preventing penetration of the substrate into the active center (the “molecular gate” is closed). Increasing the temperature will cause duplex dissociation and recovery of enzymatic activity (the “molecular gate” is opened). Two DNA–protein conjugates were obtained: the R.SsoII(S171C) conjugate with self-complementary dekadeoxyribonucleotide and dekathymidylate. The introduction of a “molecular gate” at the entry to the active center almost completely abolished the enzymatic activity at 25 °C, whereas the DNA cleavage activity of the unmodified R.SsoII(S171C) was almost 180 times higher at the same temperature. Modification by the self-complementary oligodeoxyribonucleotide seems to be particularly effective to construct a thermo-switchable enzyme. Nevertheless, non-self-complementary oligodeoxyribonucleotide of the same length can also act as “molecular gates” decreasing the enzymatic activity. Increasing the temperature (till 45 °C) enlarges the conformational freedom of the attached oligodeoxyribonucleotides making the catalytic center more accessible for the substrate. As a result, the catalytic activity of the enzyme increased approximately 150 times. The “molecular gate” strategy can be used in vitro for controlling enzyme activity by temperature. Our approach ‒ introduced as a proof of principle ‒ can also be applied for modification of other proteins that accept their substrate in a cleft, for example, some chaperones or transmembrane channels with a pore-like entrance, thus making these proteins thermosensitive and active in a desirable range of temperatures.