Conformational, fluorescence and energy parameters of Interferon α2b with different forms of oligoribonucleotides and adenosine monophosphate

Drugs based on oligoribonucleotides from yeast RNA have a reasonably low solubility in the range of 0.5-3 mg/ml. Our previous studies have shown that ORN has anti-inflammatory properties, and ORN with D-mannitol has anti-inflammatory and antiviral properties. The mechanism of this phenomenon is unclear. D-mannitol itself does not have these activities, but increases the solubility by 1.5-2 times and stabilises the structure of the ORN, which probably leads to new biological effects. In the course of our research, we found that D-mannitol affects the binding of ORN to interferon and the thermodynamic and conformational parameters of this interaction .

We studied the ability of yeast RNA (ORN), yeast RNANa salt (ORN Na), and yeast ORN: D-mannitol complex (ORN: D-M) and AMP to effect on fluorescence quenching and conformational changes of Interferon α2b. To determine the energy parameters of protein-ligand interactions, we use isothermal titration nanocalorimetry Nano ITC.

It is showing that when using ORN and ORN: D-M quenching of the fluorescent INF were 25% and 28%, AMP and AMP: DM - 15 and 21%. Quenching INF fluorescence in the titration of ORNsNa and ORNsNa: DM was 16% and 17%, AMPNa and AMPNa: DM - 8% and 10%. INF has a life-time of 2.95 ns. When interacting with ORN and ORN: D-M INF has fluorescence time of 2.37 and 2.32 ns, respectively, AMP and AMP: D-M 2.01 and 1.92 ns. When interacting with ORNsNa and ORNsNa: D-M INF has a fluorescence time of 2.73 and 2.49 ns, respectively, AMPNa and AMPNa: D-M 2.31 and 2.43 ns. Thus, ORN, and especially ORN: D-M and AMP: D-M leads to a change in the conformational mobility of interferon α-2b by increasing the content of disordered regions. At the same time, salt analogues increase the number of structured secondary elements, such as α-helices, β turns and β antiparallel sheets and probably increase the conformational stiffness of interferon α-2b. The results of the study of enthalpy changes in the titration of interferon α-2b acid form of ORN and ORN: D-M was -63.28 kJ/mol and -96.61 kJ/mol, respectively, and for the ORNNa and ORNNa: D-M respectively 4,516 and 5,139 kJ/mol. The change in entropy when adding the ORN to interferon α-2b was -38.72 and in the case of the ORN: D-M -63.53 kJ/mol*K, respectively. The difference in entropy when adding the ORNNa to interferon α-2b was 17.05 kJ/mol*K, and the ORNNa: D-M, respectively, 17.58 kJ/mol*K. A similar pattern demonstrated when studying the change in Gibbs energy during titration of interferon α-2b with ORNORN and ORN: D-M, and it was -24.56 and -33.07 kJ/mol, respectively. And when titrated with ORNNa and its ORNNa: D-M, respectively -12.9 and -12.43 kJ/mol.

These results of studying the effects of thermodynamics of different forms of RNA and their complexes with D-mannitol in the titration of interferon α-2b may indicate different sites of binding of different forms of ORN to protein, as well as other modes of binding and various types of conformational changes in the protein.