Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study

Abstract

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged-but otherwise apparently inert-surface alters the folding free energy of a simple biomolecule. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxylalkane-coated gold surface. We find that whereas surface attachment is destabilizing at low ionic strength, it becomes stabilizing at ionic strengths above ~130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis, we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate.

Figure 1

Surface attachment can alter the folding free energies of biopolymers via several mechanisms. (Left) Attachment even to a perfectly inert (non-interacting) surface is expected to stabilize the native state via excluded volume effects that reduce the entropy of the unfolded state.^, (Center) In contrast, attachment to a charged, but otherwise non-interacting surface is predicted to stabilize the unfolded state. Specifically, if the surface and the biomolecule are, as shown, of the same charge (which is the case in the study presented here), charge repulsion will destabilized the folded state more than the unfolded state, as the folded state is more compact. An oppositely charged surface, however, likely also stabilizes the unfolded state as nonspecific adsorption of the unfolded biopolymer introduces an alternative, lower energy state.^- (Right) Finally, attachment to a surface that is not inert, i.e., a surface that forms specific hydrogen bonding or hydrophobic interactions with the biopolymer, is also expected to stabilize the unfolded state as it accommodates such interactions more readily than the relatively rigid native state. Of note, prior experimental studies have argued that DNA does not form specific interactions with the 6-mercaptohexanol coated gold surface we have employed, and thus only the first two effects are expected to contribute significantly to the thermodynamics of the system employed here.

Figure 2

We have employed urea-melts^, to determine the folding free energies of a DNA stem loop both in solution (using circular dichroism spectroscopy) and when attached to a gold surface. (Left) We have monitored the latter by measuring electron transfer from a redox tag (methylene blue) on the distal terminus of our stem-loop constructs. Specifically, we used square-wave voltammetry, which is sensitive to the increase in electron transfer efficiency that occurs when the redox tag approaches the surface. (Right) At low ionic strength the stem-loop is more stable in solution than on the surface. At higher ionic strength, conditions under which both solution-phase and surface-attached stem loops are more stable, this trend is reversed.

Figure 3

(left) At low ionic strength our DNA stem-loop is less stable on the surface than in solution, presumably due to electrostaticr epulsion from the surface, which is negatively charged at the potential we have employed. At higher ionic strength electrostatic screening increases, negating this effect. Under these conditions the excluded volume effect dominates and the surface-attached molecule becomes more stable than the equivalent molecule in solution. (right) This behavior occurs because the electric field near a charged surface in an electrolyte is a strong function of counterion concentration. At higher sodium ion concentrations the electric field falls to near zero over distances shorter than a single base pair in double-stranded DNA. At lower ionic strengths, in contrast, the electric field remains significant over distances comparable to the size of our DNA stem-loop. The folding free energies observed in solution exhibit the expectedsquare root dependence on ionic strength. We fitted the surface energies to a square root dependence; this is not theoretically justified but serves as a convenient guide to the eye. The error bars in this and the following plots are standard errors derived from replicate, independent measurements.

Figure 4

A simple theoretical model, which considers only enthalpic (electrostatic) and entropic (excluded volume) contributions to the folding free energy which contains either no fitted parameters (solid line; see text for model details), or one fitted parameter-the pcz- (dashed line) fits our observations with reasonable accuracy. This, in turn, suggests that our knowledge of these effects (including prior theoretical estimates of the magnitude of the excluded volume effect) is reasonably complete and accurate and that any non-specific interactions between the DNA and our SAM-coated gold surface are negligible. The gre bar, which represents ±1 kT, is shown for scale.