Detergent properties influence the stability of the glycophorin A transmembrane helix dimer in lysophosphatidylcholine micelles

Abstract

Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important.

Figure 1

Far UV-CD spectra of the GpA TM domain in 20 mM lyso-PCs. C10-lysoPC (black), C11-lysoPC (red), C12-lysoPC (green), C13-lysoPC (blue), C14-lysoPC (cyan), C15-lysoPC (magenta), and C16-lysoPC (yellow). The measured ellipticities were converted to molar ellipticity as described in Materials and Methods.

Figure 2

Self-association of GpA peptides in 20 mM lyso-PCs. Fluorescence emission was measured in micelles with Fl-labeled peptide alone as well as with the Fl- and TAMRA-labeled peptide pair (1:1 ratio). Energy transfer was calculated from the Fl-fluorescence decrease at 525 nm. (A) FRET spectra recorded for Fl- and TAMRA-labeled peptides dissolved in 20 mM C_n lyso-PC detergents. (Upper spectrum) This data originates from peptides dissolved in C10-lyso PC micelles and the others were measured in lyso-PCs having increasing acyl-chain lengths (C10–C16), resulting in decreasing energy transfer. (B) Fraction dimer plotted against the acyl-chain length of the various lyso-PCs.

Figure 3

Concentration-dependent dimerization of the GpA TM domain. (A) GpA TM helix dimer fractions calculated from FRET spectra obtained at various C10 (●) and C16 (■) lyso-PC concentrations. The lyso-PC concentrations are given on the x axis. (Inset) FRET spectra recorded for Fl- and TAMRA-labeled peptides (1:1 mol ratio) in C10 lyso-PC micelles. All spectra were normalized at 525 nm. (Arrow) Spectral shifts at increasing C10 lyso-PC concentrations. (B) Apparent GpA TM dissociation constant determined in C10 (●) and C16 (■) lyso-PC micelles at increasing detergent concentrations. (C) Apparent dissociation free energy values (ΔG°) calculated from the apparent K_D values shown in panel A and summarized in Table S1.

Figure 4

Aggregation numbers and hydrodynamic radii of lyso-PC micelles. (A) Mean aggregation number N_agg as a function of the lyso-PC acyl-chain length at 20 mM detergent concentration determined by fluorescence quenching (●) and SLS (○). (B) Hydrodynamic radii R_h of lyso-PC micelles determined by DLS.

Figure 5

Static light-scattering analysis. Absolute light scattering intensity for the various lyso-PC at c = 20 mM in an Ornstein-Zernicke presentation for the determination of the molar masses using the intercept of an extrapolation (q → 0) in the linear regime.

Figure 6

Stoichiometry of GpA association. FRET efficiencies as a function of acceptor mole fraction are shown for 1 mM C14 lyso-PC. The total peptide and detergent concentrations were kept constant, whereas the ratio of acceptor and donor peptide varied from 0.2 to 0.85. The linear dependence of the FRET efficiency on the acceptor mole ratio demonstrates exclusive dimer formation.

Figure 7

FRET competition assay. (A) FRET pair emission spectra in 5 mM C12 lyso-PC. Addition of 0.4–2 μM unlabeled GpA TM peptide (compare panel A with panel B) results in reduced sensitized acceptor emission. Spectra were normalized at 525 nm. (Upper spectrum) This data originates from peptides in the absence of unlabeled peptide. Stepwise addition of increasing amounts of unlabeled peptide results in decreased energy transfer, i.e., in decreased fluorescence emission at 575 nm. (B) FRET efficiencies calculated from the spectra shown in panel A.

Figure 8

GpA stability and lyso-PC aggregation numbers. The fractions of dimeric GpA are plotted as a function of the lyso-PC aggregation number (different acyl-chain length) as determined by light scattering at 20-mM detergent concentration (compare to Fig. 5). The N_agg value linearly correlates with the decrease of GpA TM domain dimer fraction with an R² of 0.9.