Structure determination of a membrane protein in proteoliposomes

Abstract

An NMR method for determining the three-dimensional structures of membrane proteins in proteoliposomes is demonstrated by determining the structure of MerFt, the 60-residue helix-loop-helix integral membrane core of the 81-residue mercury transporter MerF. The method merges elements of oriented sample (OS) solid-state NMR and magic angle spinning (MAS) solid-state NMR techniques to measure orientation restraints relative to a single external axis (the bilayer normal) from individual residues in a uniformly (13)C/(15)N labeled protein in unoriented liquid crystalline phospholipid bilayers. The method relies on the fast (>10(5) Hz) rotational diffusion of membrane proteins in bilayers to average the static chemical shift anisotropy and heteronuclear dipole-dipole coupling powder patterns to axially symmetric powder patterns with reduced frequency spans. The frequency associated with the parallel edge of such motionally averaged powder patterns is exactly the same as that measured from the single line resonance in the spectrum of a stationary sample that is macroscopically aligned parallel to the direction of the applied magnetic field. All data are collected on unoriented samples undergoing MAS. Averaging of the homonuclear (13)C/(13)C dipolar couplings, by MAS of the sample, enables the use of uniformly (13)C/(15)N labeled proteins, which provides enhanced sensitivity through direct (13)C detection as well as the use of multidimensional MAS solid-state NMR methods for resolving and assigning resonances. The unique feature of this method is the measurement of orientation restraints that enable the protein structure and orientation to be determined in unoriented proteoliposomes.

Figure 1

SDS–PAGE of purified MerFt: (M) molecular weight marker, (lane 1) inclusion bodies isolated from bacteria expressing the target protein as a fusion protein with KSI, (lane 2) purified protein after Ni–NTA affinity and size exclusion chromatographies. The arrows mark the positions of the designated polypeptides.

Figure 2

Schematic timing diagrams for the pulse sequences used to obtain multidimensional SLF spectra with ¹³C detection: (A, C) three-dimensional SLF experiments that correlate ¹⁵N and ¹³C isotropic chemical shift frequencies with ¹H–¹⁵N DC and ¹H–¹³C DC frequencies, respectively, (B) three-dimensional SLF experiment that correlates ¹H–¹⁵N DC and ¹H–¹³C DC frequencies with the ¹³C iso-tropic chemical shift frequency.

Figure 3

¹³C solid-state NMR spectra of uniformly ¹³C/¹⁵N labeled MerFt in DMPC proteoliposomes. The majority of resonance intensity centered near 175 ppm is from ¹³CO backbone sites. (A, B) Spectra simulated for a single ¹³CO group in a transmembrane helix undergoing rotational diffusion around the lipid bilayer normal. The family of sidebands in panel B (red) would be observed under MAS. (C, D) Spectra obtained for a stationary sample, at 25 °C, where the protein undergoes fast rotational diffusion about the phospholipid bilayer normal, or at 10 °C, where the protein is immobile on the time scale of the static ¹³CO CSA powder pattern (~10⁵ Hz). (E, F) Spectra obtained from a sample undergoing slow (5 kHz) MAS, at 25 °C, where the ¹³CO CSA powder pattern is motionally averaged, or at 10 °C, where a family of sidebands spanning the width of the static ¹³CO CSA powder pattern is observed in the absence of protein rotational diffusion. Comparisons of the powder pattern frequency breadth (A versus B; C versus D) or the presence of spinning sidebands (E versus F) are diagnostic for the presence of fast rotational diffusion of the protein under the experimental conditions used to measure the CSA and DC powder patterns.

Figure 4

Two-dimensional MAS spectra of uniformly ¹³C/¹⁵N labeled MerFt in DMPC proteoliposomes at 25 °C: (A) homonuclear ¹³C/¹³C spin-exchange correlation spectrum, (B) heteronuclear ¹³C/¹⁵N correlation spectrum. The experiments were performed at 750 MHz under 11.11 kHz MAS.

Figure 5

Representative strip plots for residues G32–A38 of uniformly ¹³C/¹⁵N labeled MerFt in DMPC proteoliposomes at 25 °C. The spectral strips were extracted from MAS three-dimensional NCACO (red) and NCOCA (blue) spectra. Both experiments were performed at 750 MHz under 11.11 kHz MAS. Dashed lines have been added to guide the eye through the backbone resonance walk.

Figure 6

Examples of spectroscopic data for residue L31 obtained from MAS solid-state NMR spectra of uniformly ¹³C/¹⁵N labeled MerFt in DMPC proteoliposomes 25 °C: (A) two-dimensional ¹H–¹⁵N DC/¹³C shift SLF spectrum, (B) two-dimensional ¹H–¹⁵N DC/¹³C shift SLF spectral plane selected from a three-dimensional spectrum at an isotropic ¹⁵N chemical shift frequency of 118.6 ppm, (C) two-dimensional ¹H–¹³CA DC/¹³C shift SLF spectral plane selected from a three-dimensional spectrum at an isotropic ¹⁵N chemical shift frequency of 118.6 ppm, (D) two-dimensional ¹H–¹³CA DC/¹H–¹⁵N DC SLF spectral plane selected from a three-dimensional spectrum at an isotropic ¹³C chemical shift frequency of 54.6 ppm. All three spectral planes are associated with residue L31. The dashed line traces the correlation among the frequencies, which were obtained from three separate experiments using the pulse sequences diagrammed in Figure 2. The DC frequencies in the spectra correspond to the perpendicular edge frequencies of the corresponding powder patterns. Panel B shows that the ¹H–¹⁵N DC motionally averaged powder pattern for L31 has a perpendicular edge frequency of 4.7 kHz, corresponding to a splitting of 9.4 kHz, and a DC value of 18.8 kHz.

Figure 7

Examples of spectroscopic data for residue L31 obtained from MAS solid-state NMR spectra of uniformly ¹³C/¹⁵N labeled MerFt in DMPC proteoliposomes at 25 °C: (A–C) simulated powder patterns for a static peptide bond corresponding to (A) ¹⁵N amide CSA, (B) ¹H–¹⁵N DC, and (C) ¹H–¹³CA DC, (D–F) experimentally measured powder patterns recoupled under MAS. The dashed vertical lines mark the parallel (∥) and perpendicular (⊥) edge frequencies. Values of the ¹H–¹⁵N DC and ¹H–¹³CA DC measured from the perpendicular edge frequencies were multiplied by 4 to obtain the full DC values corresponding to twice the parallel edge frequency. For the ¹⁵N CSA, the perpendicular edge frequency was reduced to its traceless value and then multiplied by 2 to obtain the full CSA value.

Figure 8

Dipolar waves obtained by plotting measured values of the ¹H–¹⁵N DC versus residue number for MerFt: (A) data from MerFt in magnetically aligned bicelles, directly measured from single resonance lines in OS solid-state NMR spectra, (B) data from MerFt in un-oriented proteoliposomes, measured from motionally averaged powder patterns, recoupled under MAS. Sinusoidal fits (cyan) to the data trace the two transmembrane α-helices of the protein. (A) Reprinted from ref 12. Copyright 2006 American Chemical Society.

Figure 9

Three-dimensional structure of MerFt in DMPC phospholipid bilayers calculated from the experimental data. Both the protein structure and orientation are determined in the frame of the lipid bilayer membrane (gray) defined by the bilayer normal (n) in these calculations. The average pairwise rmsd for the 20 lowest energy structures is 1.2 Å for backbone atoms and 2.0 Å for all non-hydrogen atoms.

Figure 10

Correlations between observed and back-calculated values of orientation restraints used to calculate the structure of MerFt in phospholipid bilayers. (A) Calculated values were obtained from the initial Rosetta structural model. (B–D) Calculated values were obtained from the final refined structure. The R² correlation coefficient is listed for each type of restraint.