Folding Thermodynamics of the Hybrid-1 Type Intramolecular Human Telomeric GQuadruplex

Guanine-rich DNA sequences that may form G-quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G-quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization of G-quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G-rich sequences in the presence or the absence of their complementary C-rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26-meric human telomeric sequence d[A3G3(T2AG3)3A2]. In the presence of K1 ions, the latter adopts the hybrid-1 G-quadruplex conformation, a tightly packed structure with an unusually small number of solvent-exposed atomic groups. The K1-induced folding of the G-quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G-quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G-quadruplex formation. Based on our volume data, 432619 water molecules become released to the bulk upon the G-quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute–solvent interactions all over the surface of the folded structure. VC 2013 Wiley Periodicals, Inc. Biopolymers 101: 216–227, 2014. Keywords: G-quadruplexes; conformational transitions; volume; compressibility; expansibilit

Ionic Effects on VEGF G‑Quadruplex Stability

In a potassium solution, a modified 22-meric DNA sequence Pu22-T12T13 from a region proximal to the transcription initiation site of the human VEGF gene adopts a single parallel-stranded G-quadruplex conformation with a 1:4:1 loop-size arrangement. We measured the thermal stability, <i>T</i>_M, of the K⁺-stabilized Pu22-T12T13 G-quadruplex as a function of stabilizing K⁺ ions and nonstabilizing Cs⁺ and TMA⁺ ions. The thermal stability, <i>T</i>_M, of the Pu22-T12T13 G-quadruplex increases with the concentration of the stabilizing potassium ions, while it sharply decreases upon the addition of the nonstabilizing cations. We interpret these results as underscoring the opposing effects of internal binding and counterion condensation on the stability of the Pu22-T12T13 G-quadruplex. While centrally bound ions stabilize the G-quadruplex conformation, counterion condensation destabilizes it, favoring the coil conformation. From the initial slopes of the dependences of <i>T</i>_M on the concentration of Cs⁺ and TMA⁺ cations, we estimate that the deleterious effect of counterion condensation stems from roughly one extra counterion associated with the coil relative to the G-quadruplex state of Pu22-T12T13. The reduced accumulation of counterions around the G-quadruplex state of Pu22-T12T13 relative to its coil state is due to the low surface charge density of the G-quadruplex reflecting its structural characteristics. On the basis of the analysis of our data along with the results of a previous study, we propose that the differential effect of internally (stabilizing) and externally (destabilizing) bound cations may be a general feature of parallel intramolecular G-quadruplexes

Single-Molecule Analysis of the Supramolecular Organization of the M₂ Muscarinic Receptor and the Gα_i1 Protein

G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M₂ muscarinic receptor (M₂R) and G_i1 by single-particle photobleaching of immobilized complexes. The method was calibrated with multiplexed controls comprising 1–4 copies of fused eGFP. The photobleaching patterns of eGFP-M₂R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-G_i1 were indicative of a hexamer and unaffected by GTPγS. A complex of M₂R and G_i1 was tetrameric in both, and activation by a full agonist plus GTPγS reduced the oligomeric size of G_i1 without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-Gα_i1 by the receptor-mimic mastoparan plus GTPγS, and constitutively active eGFP-Gα_i1 was predominantly dimeric. The oligomeric nature of G_i1 in live CHO cells was demonstrated by means of Förster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled Gα_i1; stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M₂R and holo-G_i1 is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of G_i1. The structural feasibility of such a complex was demonstrated in molecular dynamics simulations