Polyethylene glycol binding alters human telomere G-quadruplex structure by conformational selection

Abstract

Polyethylene glycols (PEGs) are widely used to perturb the conformations of nucleic acids, including G-quadruplexes. The mechanism by which PEG alters G-quadruplex conformation is poorly understood. We describe here studies designed to determine how PEG and other co-solutes affect the conformation of the human telomeric quadruplex. Osmotic stress studies using acetonitrile and ethylene glycol show that conversion of the 'hybrid' conformation to an all-parallel 'propeller' conformation is accompanied by the release of about 17 water molecules per quadruplex and is energetically unfavorable in pure aqueous solutions. Sedimentation velocity experiments show that the propeller form is hydrodynamically larger than hybrid forms, ruling out a crowding mechanism for the conversion by PEG. PEGs do not alter water activity sufficiently to perturb quadruplex hydration by osmotic stress. PEG titration experiments are most consistent with a conformational selection mechanism in which PEG binds more strongly to the propeller conformation, and binding is coupled to the conformational transition between forms. Molecular dynamics simulations show that PEG binding to the propeller form is sterically feasible and energetically favorable. We conclude that PEG does not act by crowding and is a poor mimic of the intranuclear environment, keeping open the question of the physiologically relevant quadruplex conformation.

Figure 1.

Conformational change of hTel22 driven by decreased water activity in the presence of potassium at 20°C. (A) CD spectra obtained as a function of acetonitrile concentration. Addition of acetonitrile causes a shift in the CD spectrum from a major peak at 290 nm to a major peak at 265 nm. (B) The conformational change of the quadruplex monitored at 290 nm (circles) and 265 nm (solid squares) as a function of the natural logarithm of the water activity. The 295 nm signal monitors the loss of initial hybrid structure while the 265 nm signal arises from the appearance of parallel quadruplex. (C) Data for acetonitrile (black) and EG (red) plotted according to Equation (5) for determination of K_eq,0 and Δn_w. Global best-fit line (shown in red) for acetonitrile and EG was determined to be ln(K_eq) = −1.91 − 17.2ln(α_w).

Figure 2.

Isothermal titrations of hTel22 with a series of increasing MW PEGs. Colors correspond to EG (black), diEG (red), triEG (green), PEG 200 (blue), PEG 400 (cyan), PEG 600 (magenta), PEG 1000 (yellow), PEG 1500 (dark yellow), PEG 3350 (dark blue), PEG 8000 (purple, and PEG 10 000 (wine). (A) Comparison of hTel22 CD spectra obtained at the titration end point. These spectra demonstrate that all glycols cause conversion to a parallel conformation. (B) CD titration curves for each glycol studied. Arrow indicates the direction of increasing MW.

Figure 3.

(A) Transformed titration curves obtained from the primary data shown in Figure 2B. (B) Change in the transition midpoint as a function of the MW of PEG. (C) M-value plot (see text) for the conversion of the hTel22 demonstrates an increase in magnitude suggesting preferential interactions significantly contribute to the conversion of the human telomeric G-quadruplex. M-value averages and standard deviations were obtained from the analysis of multiple wavelengths. PEG 400 data were analyzed using the first half of the titration owing to non-linearity at high PEG 400 concentrations.

Figure 4.

Conformational selection by differential binding of PEG to the propeller quadruplex. (A) Representative fit of titration data obtained for PEG600 to Equation (7) (squares, 265 nm; circles, 290 nm). Optimized parameters were K_eq,0 = 0.147, n = 4 and K_B = 0.41 ± 0.01 M for the binding affinity of PEG 600. (B) Molecular dynamics shows PEG 600 interacts favorably with the planar G-tetrad faces of the propeller structure.