NMR in Bilayer Membranes

When the lipid bilayers are oriented with their surface perpendicular to the magnetic field, the solid-state NMR spectra of the membrane-associated proteins trace out maps of their structure and orientation within the membrane, and thus provide very useful structural information prior to complete structure determination (Marassi and Opella 2000; Wang et al. 2000; Marassi 2001). For example, helices give characteristic solid-state NMR spectra where the resonances from amide sites in the protein trace-out helical wheels that contain information regarding helix tilt and rotation within the membrane. Typically, trans-membrane helices have PISEMA spectra with 1SN chemical shifts between 150 and 200 ppm, and 1H-1SN dipolar couplings between 2 and 10 kHz, while helices that bind parallel to the membrane surface have spectra with shifts between 70 and 100 ppm and couplings between 0 and 5 kHz. We refer to these as the trans-membrane and in-plane regions of the PISEMA spectrum, respectively.

Glass-supported oriented phospholipid bilayers containing membrane proteins accomplish the principal requirements of immobilizing and orienting the protein for solid-state NMR structure determination. The planar lipid bilayers are supported on glass slides, and are oriented in the NMR probe so that the bilayer normal is parallel to the field of the magnet, as shown in Fig. 2.4a. The choice of lipid can be used to control the lateral spacing between neighboring phospholipid molecules as well as the vertical spacing between bilayers. The use of phospholipids with unsaturated chains leads to more expanded and fluid bilayers, and the addition of negatively charged lipids increases inter-bilayer repulsions leading to larger interstitial water layers between bilayer leaflets.

Samples of membrane proteins in lipid bilayers oriented on glass slides can be prepared by deposition from organic solvents followed by evaporation and lipid hydration, or by fusion of reconstituted unilamellar lipid vesicles with the glass surface (Marassi 2002). The choice of solvents in the first method, and of detergents in the second, is critical for obtaining highly oriented lipid bilayer preparations. In all cases the thinnest available glass slides are utilized to obtain the best filling factor in the coil of the probe. With carefully prepared samples it is possible to obtain 1SN resonance linewidths of less than 3 ppm (Marassi et al. 1997). Notably, these linewidths are less than those typically observed in single crystals of peptides, demonstrating that the proteins in the bilayers are very highly oriented, with mosaic spreads ofless than about 2°.

Bcl-XL and tBid in Bilayers

To examine the conformations of Bcl-XL and tBid associated with membranes, we obtained one-dimensional 1SN chemical shift and two-dimensional 1H/1SN PISEMA solid-state NMR spectra of the 15N-labeled proteins reconstituted in lipid bilayers (Franzin et al. 2004; Gong et al. 2004). In these samples, the lipid composition of 60% DOPC and 40% DOPG was chosen to mimic the highly nega tive charge of mitochondrial membranes. This lipid composition is identical to that of the liposomes used for the measurement of the ion channel activities of Bcl-XL, Bid, and tBid (Minn et al. 1997; Schendel et al. 1999), which were prepared in the same way as the oriented lipid bilayers used in the NMR study.

The samples of Bcl-XL and tBid in bilayers were prepared by spreading lipid vesicles, reconstituted with 15N-labeled protein, on the surface of the glass slides, allowing bulk water to evaporate, and incubating the sample in a water-saturated atmosphere (Franzin et al. 2004; Gong et al. 2004). Each sample was wrapped in parafilm and then sealed in thin polyethylene film prior to insertion in the NMR probe. The degree of phospholipid bilayer alignment can be assessed with solid-state 31P NMR spectroscopy of the lipid phosphate headgroup. The 31P NMR spectra obtained for lipid bilayers with Bcl-XL are characteristic of a liquid-crystalline bilayer arrangement, in both oriented (Fig. 2.4b) and unoriented samples (Fig. 2.4c). The spectrum from the oriented sample has a single peak near 30 ppm, as expected for highly oriented bilayers.

Membrane-Associated Bcl-xL

The spectra in Fig. 2.4 were obtained from samples of uniformly 15N-labeled Bcl-xL in oriented and unoriented lipid bilayers (Franzin et al. 2004). The spectrum obtained from oriented Bcl-xL (Fig. 2.4d) is separated into discernable resonances with distinct intensities near 80 and 170 ppm. These spectral features reflect a structural model where the helices of Bcl-XL associate with the membrane surface with limited transmembrane helix insertion. The spectrum from unoriented bilayers (Fig. 2.4e) provides no resolution among resonances, but it provides an indication of protein dynamics, because of the pronounced effects of motional averaging on such spectra. Most of the backbone sites are structured and immobile on the time-scale of the 1SN chemical shift interaction (10 kHz), contributing to the characteristic amide powder pattern between 220 and 60 ppm. Some of the Bcl-XL backbone sites, probably near the termini and loop regions, are mobile, and give rise to the resonance band centered near 120 ppm. Therefore, while certain resonances near 120 ppm, in the spectrum of oriented Bcl-XL, may reflect specific orientations of their corresponding sites, some others arise from mobile backbone sites. The intensity near 35 ppm, also present in the spectrum from the oriented sample, is from the protein amino groups, which have a considerably narrower 1SN chemical shift anisotropy. Taken together, the 1SN and 31P spectra provide evidence that Bcl-xL, an anti-apoptotic Bcl-2 family protein, associates predominantly with the membrane surface, without disruption of the membrane integrity.

2.5.1.2 Membrane-Associated tBid

The 1SN chemical shift spectrum of tBid in spherical lipid bilayer vesicles is a powder pattern (Fig. 2.5a, solid line) that spans the full range (60-220 ppm) of the

Protein Fingerprint Shift 15n

Fig. 2.4. Effect of sample orientation on the solid-state NMR spectra of isotopically labeled proteins. (A) The glass-supported phospholipid bilayer samples are oriented in the NMR probe so that the bi layer normal is parallel to the direction of the magnetic field (Bo). (B) Oriented phospholipid bilayers give single-line one-dimensional31P chemical shift NMR spectra, while (C) spherical lipid bilayer vesicles give powder patterns. (D) The one-dimensional 15N chemical shift NMR spectrum of uniformly 15N-labeled Bcl-XL in oriented lipid bilayers displays multiple resonances, compared to (E) the powder pattern that is obtained for the same protein in unoriented lipid bilayer vesicles. The 15N chemical shifts are referenced to 0 ppm for liquid ammonia

Fig. 2.4. Effect of sample orientation on the solid-state NMR spectra of isotopically labeled proteins. (A) The glass-supported phospholipid bilayer samples are oriented in the NMR probe so that the bi layer normal is parallel to the direction of the magnetic field (Bo). (B) Oriented phospholipid bilayers give single-line one-dimensional31P chemical shift NMR spectra, while (C) spherical lipid bilayer vesicles give powder patterns. (D) The one-dimensional 15N chemical shift NMR spectrum of uniformly 15N-labeled Bcl-XL in oriented lipid bilayers displays multiple resonances, compared to (E) the powder pattern that is obtained for the same protein in unoriented lipid bilayer vesicles. The 15N chemical shifts are referenced to 0 ppm for liquid ammonia amide 1SN chemical shift interaction (Fig. 2.5a, dashed line). The absence of additional intensity at the isotropic resonance frequencies (100-130 ppm) demonstrates that the majority of amino acid sites are immobile on the time-scale of the 1SN chemical shift interaction, although it is possible that some mobile unstructured residues could not be observed by cross-polarization. The peak at 35 ppm is from the amino groups at the N-terminus and sidechains of the protein. The spectrum of tBid in planar oriented lipid bilayers is very different (Fig. 2.5c). All of the amide resonances are centered at a frequency associated with NH bonds in helices parallel to the membrane surface (80 ppm), while no intensity is observed at frequencies associated with NH bonds in trans-membrane helices (200 ppm). The NMR data show no evidence of conformational exchange on the millisecond to second time-scales of the channel opening and closing events, thus eliminating the possibility of transient insertion of tBid in the membrane. Thus tBid binds strongly to the membrane surface and adopts a unique conformation and orientation in the presence of phospholipids (Gong et al. 2004).

Amide hydrogen exchange rates are useful for identifying residues that are involved in hydrogen bonding, and that are exposed to water. Typically, the amide hydrogens in trans-membrane helices have very slow exchange rates due to their strong hydrogen bonds in the low dielectric of the lipid bilayer environment, and their 15N chemical shift NMR signals persist for days after exposure to D20 (Franzin et al. 2004). Trans-membrane helices that are in contact with water because they participate in channel pore formation, and other water-exposed helical region proteins, have faster exchange rates, and their NMR signals disappear on the order ofhours (Tian et al. 2003). To examine the amide hydrogen exchange rates for membrane-bound tBid, we obtained solid-state NMR spectra after exposing the oriented lipid bilayer sample to D20 for 2h, 5h, and finally for 7 h. The majority of resonances in the 1SN chemical shift spectrum of tBid disappeared within 8 h, indicating that the amide hydrogens exchange and hence are in contact with the bilayer interstitial water.

The tBid amino acid sequence has four Lys residues (Lysl44, Lysl46, Lysl57, and Lysl58) all located in or near helix-6, one of the two helices thought to insert in the membrane and form the tBid ion-conducting pore. The spectrum of 1SN-Lys labeled tBid in bilayers is notable because its amide resonances all have chemical shifts near 80 ppm, in the in-plane region of the spectrum, and this cannot be reconciled with membrane insertion (Fig. 2.5b). Since tBid maintains a helical fold in lipid micelles and it is reasonable to assume that the helix boundaries are not changed from those of full-length Bid, the solid-state NMR data demonstrate that helix-6 does not insert through the membrane but associates parallel to its surface. This is also supported by a recent EPR study (Oh et al. 2004).

Fig.2.5. One-dimensional 15N chemical shift spectra of tBid in lipid bilayers. (a) Uniformly 15N-labeled tBid in unorlented lipid bilayer vesicles (solidline), and powder pattern calculated for a rigid 15N amide site (dot-tedline). (b) One-dimensional 15N spectrum ofselectlvely 15N-Lys-labeled tBid in oriented lipid bilayers. (c) One-dimensional 15N spectrum of uniformly 15N-labeled tBid in oriented lipid bilayers

250 200 150 100 50 0

15N chemical shift (ppm)

Fig.2.5. One-dimensional 15N chemical shift spectra of tBid in lipid bilayers. (a) Uniformly 15N-labeled tBid in unorlented lipid bilayer vesicles (solidline), and powder pattern calculated for a rigid 15N amide site (dot-tedline). (b) One-dimensional 15N spectrum ofselectlvely 15N-Lys-labeled tBid in oriented lipid bilayers. (c) One-dimensional 15N spectrum of uniformly 15N-labeled tBid in oriented lipid bilayers

250 200 150 100 50 0

15N chemical shift (ppm)

Determining the Structures of Proteins in Bilayers

When membrane proteins are incorporated in planar lipid bilayers that are oriented in the field of the NMR magnet, the frequencies measured in their multidimensional solid-state NMR spectra contain orientation-dependent information that can be used for structure determination (Marassi 2002). The PISEMA (polarization inversion with spin exchange at the magic angle) experiment gives highresolution, two-dimensional, 1H-1SN dipolar coupling / 1SN chemical shift correlation spectra of oriented membrane proteins where the individual resonances contain orientation restraints for structure determination (Wu et al. 1994). PISEMA spectra of membrane proteins in oriented lipid bilayers also provide sensitive indices of protein secondary structure and topology because they exhibit characteristic wheel-like patterns of resonances, called Pisa wheels, that reflect helical wheel projections (Schiffer and Edmunson 1967) of residues in both a-helices and P-sheets (Marassi and Opella 2000; Wang et al. 2000; Marassi 2001). When a Pisa wheel is observed, no assignments are needed to determine the tilt of a helix, and a single resonance assignment is sufficient to determine the helix rotation in the membrane. This information is extremely useful for determining the supramo-lecular architectures of membrane proteins and their assemblies.

The shape and position of the Pisa wheel in the spectrum depends on the protein secondary structure and its orientation relative to the lipid bilayer surface, as well as the amide N-H bond length and the magnitudes and orientations of the principal elements of the amide 1SN chemical shift tensor. This direct relationship between spectrum and structure makes it possible to calculate solid-state NMR spectra for specific structural models of proteins, and provides the basis for a method of backbone structure determination from a limited set of uniformly and selectively 15N-labeled samples (Marassi and Opella 2002; Marassi and Opella 2003).

The Pisa wheels calculated for single helices or strands, oriented at varying degrees in a lipid bilayer, are shown in Fig. 2.6. When the helices or strands cross the membrane with their long axes exactly parallel to the lipid bilayer normal and to the magnetic field direction (0°), all of the amide sites in each structure have an identical orientation relative to the direction of the applied magnetic field, and therefore all of the resonances overlap with the same dipolar coupling and chemical shift frequencies. Tilting the helix or strand away from the membrane normal introduces variations in the orientations of the amide NH bond vectors in the magnetic field, and leads to dispersion of the 1H-1SN dipolar coupling and 1SN chemical shift frequencies, manifest in the appearance of Pisa wheel resonance patterns in the spectra. Since helices and strands yield clearly different resonance patterns, with circular wheels for helices and twisted wheels for strands, these spectra represent signatures of secondary structure (Marassi 2001). The spectra also demonstrate that it is possible to determine the tilt of a helix or strand in lipid bilayers without resonance assignments. Pisa wheels have been observed in the PISEMA spectra of many uniformly 1SN labeled a-helical membrane proteins (Opella et al. 1999, Marassi et al. 2000; Wang et al. 2001; Marassi and Opella 2003; Park et al. 2003; Zeri et al. 2003).

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Fig.2.6. Helices and strands in oriented planar lipid bilayers give characteristic solid-state NMR spectra called Pisa wheels. The 1H-15N dipolar coupling /15N chemical shift PISEMAspectra were calculated for (a) an ideal a-helix with uniform dihedral angles =-65/-40°), and (b) an ideal p-strand with uniform dihedral angles =-135/140°), at different tilts relative to the magnetic field direction and the membrane normal. The 15N chemical shifts are referenced to 0 ppm for liquid ammonia. Spectra were calculated as described by Marassi 2001

Conformation oftBid in Lipid Bilayers

The two-dimensional 1H/1SN PISEMA spectrum of tBid in bilayers is shown in Fig. 2.7 (Gong et al. 2004). Each amide site in the protein contributes one correlation peak, characterized by 1H-1SN dipolar coupling and 1SN chemical shift frequencies that reflect the NH bond orientation relative to the membrane. For tBid, the circular wheel-like pattern of resonances in the spectral region bounded by 0-5 kHz and 70-90 ppm, provides definitive evidence that tBid associates with the membrane as surface-bound helices without trans-membrane insertion. The substantial peak overlap reflects a similar orientation of the tBid helices parallel to the membrane, and spectral resolution in this region requires three-dimensional correlation spectroscopy and selective isotopic labeling (Marassi et al. 2000).

As shown in Fig. 2.6, the NMR frequencies directly reflect the angles between individual bonds and the direction of the applied magnetic field, and, therefore, it is possible to calculate solid-state NMR spectra for specific models of proteins in oriented samples. A comparison of the calculated and experimental spectra then provides useful structural information prior to complete structure determination, which requires sequential assignment of the resonances. The spectra calculated for several orientations of an ideal 18-residue helix, with 3.6 residues per turn and identical backbone dihedral angles for all residues ^ = -57°, -47°), are shown in Fig. 2.8. This analysis demonstrates that trans-membrane helices, with orientations between 90° and 45°, have wheel-like spectra in a completely unpopulated region of the tBid spectrum. Based on a comparison of the calculated spectra with the PISEMA spectrum of tBid in lipid bilayers we place the helices of tBid nearly parallel to the lipid bilayer plane (0° orientation), with a tilt of no more than 20° from the membrane surface.

Tbid Protein
Fig.2.7. Two-dimensional 1H/15N PISEMA spectrum ofuniformly 15N-labeled tBid in oriented lipid bilayers
Solid State Nmr Pisema

Fig. 2.8. Two-dimensional solid-state NMR 1H/15N PISEMA spectrum of uniformly 15N-labeled tBid In oriented lipid bilayers. The experimental spectrum (inset box) Is compared with the spectra calculated for an 18-resldue a-hellx, with uniform backbone dihedral angles ($ = -57°; ^ = -47°), and different helix tilts (0 to 75°) relative to the membrane, depicted In the cartoon above the spectra.The 0° orientation is for a helix parallel to the membrane surface

Fig. 2.8. Two-dimensional solid-state NMR 1H/15N PISEMA spectrum of uniformly 15N-labeled tBid In oriented lipid bilayers. The experimental spectrum (inset box) Is compared with the spectra calculated for an 18-resldue a-hellx, with uniform backbone dihedral angles ($ = -57°; ^ = -47°), and different helix tilts (0 to 75°) relative to the membrane, depicted In the cartoon above the spectra.The 0° orientation is for a helix parallel to the membrane surface

Solution and solid-state NMR studies demonstrate that tBid adopts a unique helical fold in lipid environments, and that it binds the membrane without insertion of its helices. Solid -state NMR studies of the anti-apoptotic Bcl-2 family member, Bc1-Xl, also indicate that membrane insertion of the Bc1-Xl helices is only partial (Franzin et al. 2004), and solution NMR studies show that Bcl-XL adopts an extended helical conformation in lipid micelles (Losonczi et al. 2000). Both tBid and Bcl-XL form ion-conductive pores that are thought to play a role in apoptosis through their regulation of mitochondrial physiology, and it is important to note that, since the samples in both the solid-state NMR and ion channel activity studies of Bcl-XL and tBid were identical in their lipid composition and the manner of sample preparation, the membrane surface association of Bcl-XL and tBid, observed by solid-state NMR, represents the channel-active conformation of the proteins.

A model for the mode of membrane association by tBid is shown in Fig. 2.9. Cleavage by caspase-8 in the flexible loop of the soluble Bid structure (Fig. 2.9a), generates the C-terminal product tBid (Fig. 2.9b), which undergoes a conformational change and binds the surface of mitochondrial membranes (Fig. 2.9c). It is possible that the structure of tBid, destabilized by dissociation from the N-ter-minal fragment after caspase-8 cleavage, undergoes a conformational change, whereby it opens about the flexible loops that connect its helical segments, to an extended helical conformation which binds to the membrane surface. This would be similar to the mechanism proposed for the lipoprotein apolipophorin-III, which adopts a marginally stable helix bundle topology that allows for concerted opening of the bundle about hinged loops (Wang et al. 2002). It is notable that the Bid amino acid sequence (Pi4iRDMEKE147), at the beginning of helix-6, is similar to the conserved sequence (P95DVEKE100) that forms a short lipid recognition helix in apolipophorin-III. In Bid, this sequence forms a short loop that is perpendicular to the axis of helix-6 and solvent-exposed, while in apolipophorin-III it forms a short helix that is perpendicular to the helix bundle and at one solvent-exposed end of the molecule. This short motif is conserved in the Bid sequences from various species, suggesting that it plays a role in the protein biological function, and may constitute a lipid recognition domain for Bid similar to that of apolipophorin-III.

Pore formation by the Bcl-2 family proteins has been thought to involve translocation of the central core helices through the membrane, and the helices of both Bid and Bcl-XL are sufficiently long to span the lipid bilayer. However, their am-phipathic character is also compatible with membrane surface association, in a manner that is reminiscent of the antimicrobial polypeptides where binding of the polypeptide helices to the bacterial membrane surface is thought to transiently destabilize the membrane and change its morphology, inducing leakage of the cell contents, disruption of the electrical potential, and ultimately cell death (Boman 1995; Marassi et al. 1999, Marassi et al. 2000). It is notable that bacterial and mitochondrial membranes have very similar structures and surface charge, and that tBid is both capable of altering bilayer curvature, and of remodeling the mitochondrial membrane, which would be sufficient to cause the release of mitochondrial cytotoxic molecules. Thus, the BH3-independent mechanism of pore-formation and mitochondrial cytochrome-c release by tBid, may be similar to that of the an-

caspase-8

Fig. 2.9. Model for the association of tBid with the membrane surface, (a) Cleavage by cas-pase-8 in the flexible loop of the soluble Bid structure (Chou et al. 1999; McDonnell et al. 1999), generates the C-terminal product tBid (b), which undergoes a conformational change and binds the surface ofmitochondrial membranes (c)

caspase-8

Fig. 2.9. Model for the association of tBid with the membrane surface, (a) Cleavage by cas-pase-8 in the flexible loop of the soluble Bid structure (Chou et al. 1999; McDonnell et al. 1999), generates the C-terminal product tBid (b), which undergoes a conformational change and binds the surface ofmitochondrial membranes (c)

timicrobial polypeptides. In addition, the membrane surface association of tBid may serve to display the BH3 domain on the mitochondrial membrane surface, making it accessible for binding by other Bcl-2 family members. Although tBid does not insert in DOPC/DOPG lipid bilayers, it is possible that trans-membrane insertion may be driven by the presence of natural mitochondrial lipids, such as cardiolipin and monolysocardiolipin. It is also possible that the interactions with other Bcl-2 family proteins such as Bak and Bax, or with other non-homologous proteins such as the mitochondrial voltage-dependent anion channel, may promote insertion of the tBid helices through the mitochondrial membrane.

Acknowledgements. We thank David Cowburn, Stephen Fesik, and Gerhard Wagner, for sharing their solution NMR assignments for Bid and Bcl-XL. This research is supported by grants from the National Institutes of Health (R01GM065374), and the Department of the Army Breast Cancer Research Program (DAMD17-02-1-0313). The NMR studies utilized the Burnham Institute NMR Facility and the Biomedical Technology Resources for Solid-State NMR of Proteins at the University of California San Diego, supported by grants from the National Institutes ofHealth (P30CA30199; P41EB002031).

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