The one- and two-dimensional 1H–15N spectra of [15N]Phe-labeled OmpX in 14-O-PC/6-O-PC parallel phospholipid bicelles are shown in . The seven Phe residues in the protein sequence give seven fully resolved peaks, and each peak in the SLF spectrum provides a pair of 15N chemical shift and 1H–15N dipolar coupling frequencies that can be used as residue-specific orientation restraints for protein structure determination, provided that peak assignments are determined. Peak assignments were obtained by an iterative method of simulated annealing and back-calculation from the crystal structure of OmpX, as described below.
Figure 1 One- and two-dimensional 1H–15N spectra of [15N]Phe-labeled OmpX in 14-O-PC/6-O-PC parallel bicelles, in H2O (black) or D2O (red). (A and B) 15N chemical shift spectra. Asterisks denote peaks that disappear in D2O. (C) Experimental SLF spectrum. (more ...)
The frequencies measured for the seven Phe peaks are reported in . Although the SLF experiment does not provide direct information about the signs of the dipolar couplings, the signs can be determined from the peak positions in the spectrum. The dipolar coupling signs for peaks assigned to F90, F107, F115, F125, and F148 (−, +, +, +, and +, respectively) can be determined with a high degree of confidence since these peaks fall in well-defined regions of the SLF spectrum where the sign is unambiguous (46
). The signs for peaks assigned to F24 and F43 are less easily determined by simple inspection. They were estimated (− and −, respectively) from the spectra predicted for oriented β
) analogous to the characteristic wheel-like patterns observed for α-helices (48
), and verified by the back-calculation analysis.
Dipolar Coupling (DC) and Chemical Shift (δ) Frequencies Measured for [15N]Phe-Labeled OmpX in 14-O-PC/6-O-PC Bicelles or in Isotropic 6-O-PC Micellesa
To obtain residue-specific assignments, we first identified three peaks that disappear from the spectrum after H–D exchange (, black, #1, #4, and #7) and assigned them to three possible residues (F90, F107, and F148) by comparison with the HSQC spectra of OmpX in 6-O-PC micelles obtained in H2
O or D2
O (), using the assignments for OmpX in DHPC (1,2-dihexyl-sn
-glycero-3-phosphocholine, the phosphodiester analogue of 6-O-PC) deposited by Wuthrich and co-workers (7
) in the Biological Magnetic Resonance Data Bank (BMRB entry 4936).
Selected region of the superimposed 1H–15N HSQC spectra of OmpX in 6-O-PC micelles, in H2O (black) or D2O (red). Peaks for F24, F43, F115, and F125 persist in the D2O spectrum.
Next we generated six (3!) test assignments () for the possible permutations of three labels among three peaks, and six corresponding lists of dipolar couplings and chemical shift anisotropies, which were used in a rigid body simulated annealing calculation to orient the coordinates of the crystal structure relative to the magnetic field and to the membrane. Rigid body simulated annealing calculations for each of the six test assignments of F90, F107, and F148 were performed with an energy function that included only dipolar coupling restraints, while the chemical shift anisotropies were used as a comparison set to assess the closeness of fit to the experimental data. In this calculation, all the atoms of the minimized coordinates of OmpX were grouped as a rigid body and allowed to undergo both rotation and translation, while the coordinates of an external axis system with the Z axis parallel to the magnetic field were held fixed. The resulting oriented coordinates were used, in turn, to back-calculate the SLF spectrum, which was then evaluated for its closeness of fit to the experimental solid-state NMR spectrum by computing the rmsds between the observed and calculated NMR frequencies. To obtain comparable, frequency-independent, estimates of the rmsds for the dipolar couplings and chemical shifts, the deviations were scaled by the spectral range available for each spin interaction (20 kHz; 150 ppm). Since the chemical shifts were not implemented as working restraints during simulated annealing, their rmsd provides a measure of cross-validation for the structural analysis. The resulting back-calculated spectra and protein orientations are shown in , and the corresponding rmsds are reported in .
Figure 3 Test assignments generated for six possible ways (A–F) of distributing three Phe labels (F90, F107, and F148) among three peaks, shown in black to indicate that they disappear in D2O; peaks in red persist in D2O. For each test assignment, the (more ...)
Table 2 Root-Mean-Square Deviations between Experimental and Calculated Values of the Dipolar Coupling (DC), Chemical Shift Anisotropy (CSA), and Combined DC and CSA (TOT), Obtained for Each of the Six Test Assignments of F90, F107, and F148, shown in (more ...)
Of the six test assignments, only two () generated plausible transmembrane orientations of the OmpX β-barrel, while the others gave inconsistent and highly tilted orientations that are incompatible with membrane insertion or with the membrane hydrophobic thickness. The first test assignment () was able to reproduce the pattern of resonances in the experimental spectrum very well and gave the lowest rmsds for both dipolar coupling and chemical shift frequencies.
The frequencies obtained by back-calculation from the lowest-rmsd test assignment of F90, F107, and F148, in , were further compared to the 24 (4!) possible assignments for the four remaining peaks of residues F24, F43, F115, and F125 (Table S1
). One result with significantly lower rmsds from the experimental frequencies was accepted, and the seven dipolar couplings assigned in this way were used as working restrains in a second and final simulated annealing calculation performed with semi-rigid body dynamics, while the corresponding chemical shift anisotropies were used as a comparison set. In this step, only the backbone CA, C, N, and O atoms were grouped as a rigid body and allowed to undergo rotation and translation, and the coordinates of the external axis were fixed. The energy function included dipolar coupling restraints, as well as terms for covalent geometry, van der Waals contacts, and the torsion angle database Rama potential (37
) for selection of preferred side chain conformations relative to the backbone dihedral angles.
This final simulated annealing step yields the coordinates for oriented OmpX shown in and the corresponding back-calculated spectrum in . The results show that OmpX traverses the membrane with a modest (7.3 ± 1.6°) tilt of the barrel axis relative to the membrane normal. The 20 calculated structures have average pairwise rmsds of 0.23 Å for the backbone atoms and 1.15 Å for all non-hydrogen atoms. Within this set, the structure with the lowest energy () has backbone and non-hydrogen atom rmsds of 0.49 and 1.62 Å, respectively, from those of the original PDB file. The calculated spectrum is very similar to that obtained experimentally, and the resulting rmsds of 0.015 for the dipolar couplings, 0.028 for the chemical shifts, and 0.021 for the combined frequencies reflect experimental errors as well as uncertainties in the N–H bond length, in the Szz value of the bicelle order parameter, in the 15N chemical shift tensor, and in the atomic coordinates of the molecular structure.
Figure 4 Membrane orientation of OmpX. (A and D) OmpX after refinement against the dipolar couplings measured for the seven Phe residues. The average tilt angle (τ) between the barrel axis (b) and the membrane normal (Z) is 7.3° (±1.6°). (more ...)
Varying the order parameter Szz from 1.0 to 0.7 [corresponding to values of Da(bicelle) from 10.52 to 7.36 kHz] yields back-calculated spectra with dipolar couplings and chemical shift anisotropies that scale as Szz, as expected, but has no effect on the orientation of OmpX in the membrane (). Among these values of Szz, the best agreement between experimental and calculated frequencies was obtained for Szz = 0.8 and Da(bicelle) = 8.41 kHz ().
Since the chemical shifts were not implemented as working restraints during simulated annealing, they play no role in determining the membrane orientation of OmpX, and given that a common chemical shift tensor was used for all Phe peaks in the analysis, it is remarkable that the back-calculated SLF spectrum reproduces both dipolar couplings as well as chemical shifts so well. Furthermore, using the residue-specific values of δiso, measured for each Phe peak in the HSQC spectrum of OmpX in micelles, instead of the average value of all the Phe peaks, has little effect on the back-calculated orientation-dependent chemical shifts (). The very low rmsds between the experimental and calculated spectra suggest that residue-specific tensor variations are minor compared to the spectral manifestation of molecular orientation, indicating that chemical shifts as well as dipolar couplings can be used as useful restraints for structure determination and refinement.
The oriented coordinates show that OmpX traverses the membrane with a 7° tilt of the barrel axis relative to the membrane normal. This is similar to the orientations of OmpX reported in the PDB_TM [Protein Data Bank of Transmembrane Proteins (http://pdbtm.enzim.hu/
)] and OPM [Orientations of Proteins in Membranes (http://opm.phar.umich.edu/
)] databases, where computational methods are used to position proteins in membranes. The oriented membrane protein coordinates in the OPM database are obtained by minimizing the energy involved in transferring a protein from water to a hydrophobic slab approximating the membrane hydrocarbon core (50
), while the PDBTM database orientations are derived from hydrophobicity scales of amino acid residues and estimates of nonpolar accessible surface area (52
). Lee (9
) has noted that although the fatty acyl chain lengths of the phospholipid component of the bacterial outer membrane are predominantly C16 or C18, the fatty acyl chains of the lipopolysaccharide component are mostly saturated with a length of C14 and some C12 (54
), suggesting that the thickness of the bacterial outer membrane will be similar to that of a 14-O-PC lipid bilayer, which is 23 Å, matching the hydrophobic thickness of the protein very well. Therefore, our result obtained in 14-O-PC is likely to reflect the state in the natural membrane environment.