A bifunctional spin label reports the structural topology of phospholamban in magnetically-aligned bicelles

Abstract

We have applied a bifunctional spin label and EPR spectroscopy to determine membrane protein structural topology in magnetically-aligned bicelles, using monomeric phospholamban (PLB) as a model system. Bicelles are a powerful tool for studying membrane proteins by NMR and EPR spectroscopies, where magnetic alignment yields topological constraints by resolving the anisotropic spectral properties of nuclear and electron spins. However, EPR bicelle studies are often hindered by the rotational mobility of monofunctional Cys-linked spin labels, which obscures their orientation relative to the protein backbone. The rigid and stereospecific TOAC label provides high orientational sensitivity but must be introduced via solid-phase peptide synthesis, precluding its use in large proteins. Here we show that a bifunctional methanethiosulfonate spin label attaches rigidly and stereospecifically to Cys residues at i and i+4 positions along PLB's transmembrane helix, thus providing orientational resolution similar to that of TOAC, while being applicable to larger membrane proteins for which synthesis is impractical. Computational modeling and comparison with NMR data shows that these EPR experiments provide accurate information about helix tilt relative to the membrane normal, thus establishing a robust method for determining structural topology in large membrane proteins with a substantial advantage in sensitivity over NMR.

Keywords: Bicelles; Bifunctional spin label; EPR; Molecular dynamics; Orientation; Phospholamban.

Figure 1

Left: structural formula of BSL before and after reaction. Right: energy-minimized structure of BSL attached to Cys at positions 32 and 36 on monomeric PLB, based on the NMR structure [1]. BSL dihedral angles were initialized from an x-ray crystal structure of BSL attached to a helix on T4 lysozyme [2], then further refined by molecular dynamics simulations to produce the shown structure. The bilayer normal is indicated by n, the bilayer surface by dashed lines, and dotted lines indicate the approximate boundaries of the hydrophobic interior.

Figure 2

BSL nitroxide coordinate system (x_P, y_P, z_P) with membrane normal (n) and helix long axis (h) vectors. (a) The membrane normal is described by the spherical angles θ_np and ϕ_np. (b) The helix long axis is described by the spherical angles θ_hp and ϕ_hp. (c) θ_nh is the angle spanning n to h, the PLB transmembrane helix tilt relative to the normal (d). (e) Closeup of the nitroxide coordinate system superimposed on the nitroxide structure. (f) Euler rotation (z-y-z convention) with diffusion tilt angles β_D and γ_D used to transform the principal diffusion axis (z_R = n) into the probe coordinate system shown in (a) [33].

Figure 3

EPR spectra (black) of monofunctional 32-BSL-PLB (left) and bifunctional 32/36-BSL-PLB (right) in randomly-oriented lipid vesicles at 200 K (top) and 298 K (bottom), with best fits (red) for rigid-limit (200 K) and MOMD (298 K) models (parameters from fits in Table 1). Sweep width on horizontal axis is 120 G.

Figure 4

EPR spectra (black) of monofunctional 32-BSL-PLB (left) and bifunctional 32/36-BSL-PLB (right) in bicelles with the membrane normal aligned parallel (top) and perpendicular (bottom) with respect to the applied magnetic field. Best fits (red) correspond to global analysis of (a) and (b) or (c) and (d). Parameter values from fits are in Table 1. Spectra were acquired at 298 K, and sweep width on horizontal axis is 120 G.

Figure 5

Distribution for angles between nitroxide principal axis and PLB long helix axis (θ_hp and ϕ_hp) reported by molecular dynamics simulation of BSL modeled at positions 32 and 36 of the transmembrane helix. Peak values and associated uncertainties shown as inset text.