Structure determination of a membrane protein with two trans-membrane helices in aligned phospholipid bicelles by solid-state NMR spectroscopy

Abstract

The structure of the membrane protein MerFt was determined in magnetically aligned phospholipid bicelles by solid-state NMR spectroscopy. With two trans-membrane helices and a 10-residue inter-helical loop, this truncated construct of the mercury transport membrane protein MerF has sufficient structural complexity to demonstrate the feasibility of determining the structures of polytopic membrane proteins in their native phospholipid bilayer environment under physiological conditions. PISEMA, SAMMY, and other double-resonance experiments were applied to uniformly and selectively (15)N-labeled samples to resolve and assign the backbone amide resonances and to measure the associated (15)N chemical shift and (1)H-(15)N heteronuclear dipolar coupling frequencies as orientation constraints for structure calculations. (1)H/(13)C/(15)N triple-resonance experiments were applied to selectively (13)C'- and (15)N-labeled samples to complete the resonance assignments, especially for residues in the nonhelical regions of the protein. A single resonance is observed for each labeled site in one- and two-dimensional spectra. Therefore, each residue has a unique conformation, and all protein molecules in the sample have the same three-dimensional structure and are oriented identically in planar phospholipid bilayers. Combined with the absence of significant intensity near the isotropic resonance frequency, this demonstrates that the entire protein, including the loop and terminal regions, has a well-defined, stable structure in phospholipid bilayers.

Figure 1

Kyte-Doolittle hydropathy plot and the amino acid sequences^, of MerF and MerFt.

Figure 2

One-dimensional ¹⁵N NMR spectra of aligned samples of uniformly ¹⁵N labeled MerFt. A. Mechanically aligned in 14-O-PC bilayers on glass plates. B. Magnetically aligned in 14-O-PC/6-O-PC, q=3.2, parallel bicelles. C. Magnetically aligned in perpendicular bicelles.

Figure 3

One-dimensional ¹⁵N NMR spectra of magnetically aligned samples of selectively ¹⁵N labeled MerFt in 14-O-PC/6-O-PC, q=3.2, bicelles. A. Parallel bicelles. B. Perpendicular bicelles. The samples in panel “A” were “flipped” from the perpendicular to the parallel alignment by addition of 3 mM YbCl₃. Spectra of valine (6 sites), leucine (13 sites), isoleucine (6 sites), and tyrosine (3 sites) labeled MerFt samples are shown left to right. The narrow resonance near 40 ppm in the Leucine spectra is assigned to the N-terminal amino group.

Figure 4

Two-dimensional ¹⁵N chemical shift/¹H-¹⁵N dipolar coupling NMR spectrum of uniformly ¹⁵N-labeled MerFt aligned in 14-O-PC/6-O-PC, q=3.2, perpendicular bicelles. A. Composite spectrum consisting of a PISEMA spectrum (¹⁵N Shift < 105 ppm) and a SAMMY spectrum (¹⁵N Shift > 105 ppm), as marked by the arrow on the chemical shift axis. B. Representation of the two-dimensional spectrum with the centers of the circles corresponding to the values in Table 1, which were obtained from the best available measurement. The diameter of the circles corresponds to the estimated experimental uncertainty in the measurements. The resonance assignments are noted. Colors are used to denote resonances with segments of the protein: N-terminus, residues 13–26 (grey); helix 1, residues 27–41 (red); loop, residues 42–51 (green); helix 2, residues 52–68 (blue); C terminus, residues 69–70 (purple).

Figure 5

Two-dimensional ¹⁵N chemical shift/¹H-¹⁵N dipolar coupling NMR spectra of selectively ¹⁵N-labeled MerFt samples aligned in 14-O-PC/6-O-PC, q=3.2, perpendicular bicelles. The spectra are composites derived from PISEMA and SAMMY experiments, which were individually optimized at various ¹H carrier frequencies, typically between 8 ppm and 9.5 ppm, and resulting from between 32 t₁ and 72 t₁ increments, depending upon the resolution needed to assign for each sample. Also shown are indexed, ideal PISA wheels calculated for an α-helix with uniform dihedral angles (Φ=−61°, Ψ=−45°), helix tilt of 18° and bicelle order parameter S = 0.8. The wheels are superimposed on the spectra of Ile (2 sites) and Leu labeled (13 sites) MerFt. Comparison with ideal PISA wheels gives an immediate visual estimate of the helix tilt, which here appears to be the same for both α-helices. Assignment of the only helical Phe residue, F54, by comparison with the PISA wheel is straightforward and enables indexing of the second helix. For more complex cases, like the Leu spectrum, indexing of the PISA wheels can be carried out by least-square fitting of the patterns of the selectively labeled samples, assigned by residue type. The indexed wheels give an estimate of the phase of the helices.

Figure 6

One-dimensional ¹⁵N NMR spectra of selectively ¹³C′-Tyr and ¹⁵N-Leu labeled MerFt aligned in perpendicular bicelles. A. With only ¹H decoupling. B. With both ¹H and ¹³C decoupling.

Figure 7

The assignment of the three tyrosine resonances in the spectrum of MerFt. A. Two-dimensional PISEMA spectrum of selectively ¹⁵N-Tyr labeled MerFt in perpendicular bicelles. An ideal PISA wheel calculated for an α-helix with uniform dihedral angles (Φ=−61°, Ψ=−45°), helix tilt of 18° and bicelle order parameter S=0.8 is superimposed on the spectrum. B. One-dimensional ¹⁵N NMR spectra of selectively ¹³C′-Gly,¹⁵N-Tyr labeled MerFt in perpendicular bicelles. With only ¹H decoupling (top) and with both ¹H and ¹³C decoupling (bottom).

Figure 8

Plot of experimental dipolar coupling frequencies as a function of residue number with Dipolar wave fits. The frequencies and are divided into four groups that are color-coded by reside number in the primary sequence of the peptide corresponding to the secondary structural domains of the protein: N-term residues 13–26 (grey), first helix residues 27–41 (red wave), loop residues 42–51 (green), second helix residues 52–68 (blue wave) and C-terminus (purple).

Figure 9

A. Flowchart for the structure calculations. B. Ramachandran plots (RAMA) of prolines, pre-prolines, glycines and all other residues used to restrict the space of the torsion angles for non-helical residues. C. Definitions of parameters for the helix-helix packing constraints described in the text.

Figure 10

Three-dimensional backbone structure of residues 27 – 68 of MerFt in phospholipid bilayers. A. Wire frame representation. B. Ribbon diagram. C. Back-calculated spectrum (dots) from one of the structures shown in part A. superimposed on the representation of the experimental data. D. Ramachandran plot with all of the backbone angles marked with dots.