Lipid bilayer topology of the transmembrane alpha-helix of M13 Major coat protein and bilayer polarity profile by site-directed fluorescence spectroscopy

Abstract

This article presents a new formalism to perform a quantitative fluorescence analysis using the Stokes shift of AEDANS-labeled cysteine mutants of M13 major coat protein incorporated in lipid bilayers. This site-directed fluorescence spectroscopy approach enables us to obtain the topology of the bilayer-embedded transmembrane alpha-helix from the orientation and tilt angles, and relative bilayer location. Both in pure dioleoylphosphatidylcholine and dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers, which have a similar bilayer thickness, the tilt angle of the transmembrane helix of the coat protein turns out to be 23 degrees +/- 4. Upon decreasing the hydrophobic thickness on going from dieicosenoylphosphatidylcholine to dimyristoylphosphatidylcholine, the tilt angle and orientation angle of the transmembrane alpha-helix change. The protein responds to an increase of hydrophobic stress by increasing the tilt angle so as to keep much of its hydrophobic part inside the bilayer. At the same time, the transmembrane helix rotates at its long axis so as to optimize the hydrophobic and electrostatic interactions of the C-terminal phenylalanines and lysines, respectively. The increase of tilt angle cannot completely keep the hydrophobic protein section within the bilayer, but the C-terminal part remains anchored at the acyl-chain/glycerol backbone interface at the cost of the N-terminal section. In addition, our analysis results in the profile of the dielectric constant of the hydrophobic domain of the bilayer. For all phospholipid bilayers studied the profile has a concave shape, with a value of the dielectric constant of 4.0 in the center of the bilayer. The dielectric constant increases on approaching the headgroup region with a value of 12.4 at the acyl-chain/glycerol backbone interface for the various phosphatidylcholines with different chain lengths. For dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers the value of the dielectric constant at the acyl-chain/glycerol backbone interface is 18.6. In conclusion, the consistency of our analysis shows that the applied cysteine-scanning mutagenesis method with AEDANS labeling of a helical transmembrane protein in combination with a quantitative formalism offers a reliable description of the lipid bilayer topology of the protein and bilayer properties. This also indicates that the spacer link between the protein and AEDANS label is long enough to monitor the local polarity of the lipid environment and not that of the amino-acid residues of the protein, and short enough to have the topology of the protein imposing on the fluorescence properties of the AEDANS label.

FIGURE 1

Schematic representation of an α-helix before (a) and after positioning in a bilayer including a rotation and tilt (b).

FIGURE 2

Normalized fluorescence spectra of different mutants, reconstituted in DOPC/DOPG bilayers with the AEDANS label attached at positions 22 (dashed curve), 46 (dotted curve), and 34 (solid curve) in the primary sequence.

FIGURE 3

Stokes shift Δν of AEDANS-labeled M13 coat protein mutants as a function of the amino-acid residue number n at which the label is attached in DOPC/DOPG (a) and 18:1 PC (b). Computer fits are included represented by a solid line in the data range (n = 15–46). The dashed lines represent extrapolations of the fitted function.

FIGURE 4

Schematic projection of the calculated helix in a DOPC/DOPG bilayer, showing the bilayer depth of various typical amino-acid residues, Trp-26 (W); Phe-42 and Phe-45 (F); Lys-40, Lys-43, and Lys-44 (K); and of the reference position n = 29 (#), with respect to the acyl-chain/glycerol backbone interfaces (dashed lines). For this lipid system the reference position is almost exactly facing the tilt. Positions are calculated for a model helix with a radius of 5 Å using the parameters in Table 2.

FIGURE 5

Orientation angle α (□) and tilt angle β (○) for PC bilayers from Table 3 as a function of hydrophobic thickness.

FIGURE 6

Distances of typical amino-acid residues to the bilayer center for isoleucine (32, top left) and to the acyl-chain/glycerol backbone interface for tryptophan (26, top right), phenylalanines (42 and 45, bottom left), and lysines (40, 43, and 44, bottom right) plotted as a function of the hydrophobic thickness for a model helix with a radius of 5 Å, using the parameters in Table 3.

FIGURE 7

Calculated Δν profiles (top) and ɛ(d) profiles (bottom) for bilayers of 18:1 PC (solid curves) and DOPC/DOPG (dotted curves). The ɛ(d) curves are calculated according to Eqs. 1–3 using n_r = 1.5 (Salomon et al., 2000), a = 0.36 nm, Δμ = 5.63 D (Ren et al., 1999), and C = Δν_hexane (Δf ≈ 0) = 5344 cm⁻¹ (this article). Vertical lines represent locations of the acyl-chain/glycerol backbone interface following from the published hydrophobic thicknesses d_h (Ridder et al., 2002) at d = ½d_h.