Effect of detergents on the association of the glycophorin a transmembrane helix

Abstract

We have examined the role of the environment on the interactions between transmembrane helices using, as a model system, the dimerization of the glycophorin A transmembrane helix. In this study we have focused on micellar environments and have examined a series of detergents that include a range of alkyl chain lengths, combined with ionic, zwitterionic, and nonionic headgroups. For each we have measured how the apparent equilibrium constant depends on the detergent concentration. In two detergents we also measured the thermal sensitivity of the equilibrium constant, from which we derive the van't Hoff enthalpy and entropy. We show that several simple models are inadequate for explaining our results; however, models that include the effect of detergent concentration on detergent binding are able to account for our measurements. Our analysis suggests that the effects of detergents on helix association are due to a pair of opposing effects: an enthalpic effect, which drives association as the detergent concentration is increased and which is sensitive to the chemical nature of the detergent headgroup, opposed by an entropic effect, which drives peptide dissociation as the detergent concentration is raised. Our results also indicate that the monomer-monomer interface is relatively hydrophilic and that association within detergent micelles is driven by the enthalpy change. The wide variations in glycophorin a dimmer, stability with the detergent used, together with the realization that this results from the balance between two opposing effects, suggests that detergents might be selected that drive association rather than dissociation of peptide dimers.

FIGURE 1

Effect of detergent concentration on the GpA equilibrium in detergent micelles. A and B shows the effect of the detergent concentration on the apparent GpA dimer dissociation constant. Results with two representative detergents are shown: C₁₂DAO (A) and C₁₂Sulfate (B). C shows the typical quasilinear relationships obtained for plots of apparent dissociation free energy (ΔG°) as a function of the logarithm of the micellar detergent concentration. The micellar detergent concentration is calculated by subtracting the CMC from the total detergent concentration. Data for four typical detergents are shown: C₁₂Sulfate (▪), C₁₂DAO (•), C₁₀Maltoside (+), and C₁₂Maltoside (×). Two sets of data obtained with different lots of C₁₂Sulfate are shown as solid and open symbols. The points represent best-fit values obtained by nonlinear fitting of the FRET titration data at each detergent concentration. The lines represent best fit values obtained by fitting the complete set of FRET titration data in a given detergent. The measured CMC of each detergent studied is listed in Table 1.

FIGURE 2

Effect of temperature on the thermodynamics of GpA dimerization. A and B show van't Hoff plots at three different detergent concentrations spanning two orders of magnitude: 2 mM (▴) 20 mM (•), and 91 mM (▪). The data illustrated were obtained with the detergent C₁₂DAO. At each detergent concentration, the slopes obtained from these graphs were used to determine the standard enthalpy (ΔH°) and entropy (ΔS°) changes of dissociation.

FIGURE 3

Effect of detergent concentration on thermodynamic parameters for dimerization. Data are shown for two detergents: C₁₂DAO (A) and C₁₂Sulfate (B). In each panel, circles represent standard enthalpy changes (ΔH°); squares, the contribution of the standard entropy changes to the standard free energy change (−TΔS°) at 25°C, and crosses (+) standard free energy changes (ΔG°) calculated as ΔH°−TΔS°. The second group of crosses (×) show the standard free energy changes calculated from the observed equilibrium constants at 25°C.