K⁺/Pb²⁺ ion exchange-induced structural transitions of G-quadruplexes under molecular crowding conditions

Because biochemical experiments are typically performed under dilute conditions despite the highly crowded nature of the cellular environment, crowding agents such as PEG are used to mimic intracellular conditions and investigate biomolecular stability and structural transitions. In this study, we systematically investigated the effect of molecular crowding on the ion exchange between K⁺ and Pb²⁺ in four representative G-quadruplexes, TBA, HTG, PS2.M, and T2, using PEG200 as a crowding agent. Circular dichroism (CD), fluorescence (FL), and thermal melting analyses revealed that the extent of Pb²⁺ induced conformational change and stability strongly depended on both sequence and crowding conditions. In contrast to dilute conditions, the order of structural variation by Pb²⁺ under molecular crowding shifted to TBA > HTG > PS2.M > T2. TBA exhibited the greatest conformational flexibility and Pb²⁺ affinity, whereas PS2.M showed a comparatively increased binding affinity relative to the other sequences as the degree of molecular crowding increased. These results highlight that molecular crowding modulates ion-induced structural transitions of G-quadruplexes, providing new insights into G-quadruplex behavior under physiologically relevant conditions.