Structural analysis of peptide identified from the 2KRR domain of the nucleolin protein with a c-Myc G4 structure using biophysical and biochemical methods

Abstract

For the first time, the c-Myc G4 structure is reported to be stabilized by binding of the peptide (derived from the 2KRR domain of the nucleolin protein called the Nu peptide) in the loop region of the G-quadruplex structure by stacking interactions. CD results showed the formation of parallel G4 structure in the presence of 100 mM Na⁺ or 100 mM K⁺ with the appearance of two isodichroic points at 229 nm, 254 nm and 252 nm in the presence of 100 mM Na⁺ or 100 mM K⁺, respectively. In addition, in UV thermal and CD melting studies, we observed drastic changes with an increase in the hyperchromicity at a DNA : peptide ratio of 1 : 50. On titrating the Nu peptide with c-Myc G4, we calculated the value of binding constant (K_a) by plotting fluorescence intensity and DNA concentration as 0.1369 ± 0.008 μM and 0.1277 ± 0.073 μM in Na⁺ and K⁺, respectively, which confirms the strong association of Nu peptide with c-Myc G4. The Nu peptide showed preferential cytotoxicity against MDA-MB-231 cells with IC₅₀ values of 5.020 μM and 5.501 μM after 72 and 96 hours. This approach suggests a novel strategy to target G4 structure using natural key peptide segments derived from G4 stabilizing protein.