Verification of the method
Results were verified through all available experimental data for 24 TM proteins of known 3D structure whose spatial positions in bilayers have been experimentally studied: rhodopsin (1gzm), bacteriorhodopsin (1py6), sensory rhodopsin II (1h2s), photosynthetic reaction centers from two species (1rzh and 1dxr), cytochrome c oxidase (1v55), Na+-ATPase (1yce), Ca2+-ATPase(1iwo, 2agv, 1wpe, 1t5s, 1su4, 1wpg), phospholamban (1zll), lactose permease LacY (1pv6), protein translocase SecY (1rh5), Na+/H+ antiporter (1zcd), K+-channel KcsA (1r3j), MscL mechanosensitive channel (1msl), acetylcholine receptor (2bg9), outer membrane proteins OmpA (1qjp), OmpX (1qj8), OmpLA (1qd6), OmpF (1hxx), ferric enterobactin receptor receptor FepA (1fep), ferric hydroxamate uptake receptor FhuA (1qfg), cobalamine transporter BtuB (1nqe), α-hemolysin (7ahl), and gramicidin A (1grm). A number of methods, such as chemical modification, spin-labeling, fluorescence spectroscopy, ATP FTIR, NMR, X-ray scattering, neutron diffraction, electron cryo-microscopy, and hydrophobic matching studies were used to determine hydrophobic thicknesses or tilts of these proteins, to locate their membrane-embedded segments, and to evaluate penetration depths and environments of their residues in lipid bilayers or detergents (Supplemental Material).
Comparison with experimental tilt angles
NMR studies of the bacteriorhodopsin trimer show that helix A and the extracellular section of helix B are tilted with respect to the bilayer normal by 18°–22° and by less than 5°, respectively (Kamihira et al. 2005). This is consistent with the calculated tilt angles of 23° and 5°, respectively, for these helices in a trimer (1qm8). The calculated tilt of gramicidin A channel (2° ± 10°) is also in excellent agreement with solid-state NMR and infrared dichroism studies that show a nearly perpendicular arrangement of the dimer in the membrane (Nabedryk et al. 1982; Andronesi et al. 2004; Andersen et al. 2005). The calculated average tilts of α-helices or β-strands in TM proteins correlate well with ATR FTIR spectroscopy data (Table 3). However, the experimental values are systematically larger, which could be due to some orientational disorder under the experimental conditions. It has been noted that values of τ obtained by ATR FTIR spectroscopy may represent upper limits of the actual tilt angles for α-helical peptides, due to such disorder (Bechinger et al. 1999; de Planque and Killian 2003).
Table Table 3.. Average tilt angles (°) of TM α-helices or β-strands relative to the membrane normal calculated by PPM 1.0 (βcalc) and determined by ATR FTIR spectroscopy (βexper)
The overall tilt calculated for rhodopsin (τ ∼ 8°) is consistent with the orientation of the protein in 2D crystals (Krebs et al. 2003). The tilts of seven individual helices were 32° (I), 27° (II), 27° (III), 4° (IV), 32° (V), 9° (VI), and 16° (VII) in the 2D crystals and 33° (I), 25° (II), 27° (III), 9° (IV), 15° (V), 13° (VI), and 20° (VII) in the calculated orientation of rhodopsin. Thus, a significant discrepancy was found only for TM helix V of rhodopsin, which is the least reliably defined in EM maps. The calculated orientation of hetero-trimeric SecY complex (1rh5) is similar but not identical to that in the 2D crystal where this protein forms a dimer of trimers (Breyton et al. 2002).
Comparison with membrane penetration depths of individual residues
The calculated membrane-embedded portions of the regular secondary structures agree with studies of BtuB transpoter, bacteriorhodopsin, FepA receptor, and MscL and KcsA channels by spin-labeling, MscL by fluorescence, and Na+ ATPase by EM and X-ray crystallography (Table 4). The experimental and calculated penetration depths of individual spin-labeled residues are generally consistent for MscL and KcsA channels, bacteriorhodopsin, and FepA receptor (Fig. 2). However, experimental and calculated depths may deviate up to 5 Å, as observed for residue 69 in MscL channel (Dcalc = 1.1 Å and Dexper = 6.0 Å). Such deviations probably appear because the depths were taken for the Cβ-atom of the spin labeled cysteine instead of the nitroxyl group, which actually interacts with the paramagnetic quenchers. The penetration depth of the nitroxyl radical in Cys69 of MscL can be in the range of −1.0 Å to 6.5 Å, depending on four χ angles of the spin-labeled cysteine. The latter value is more consistent with the experiment.
Figure Figure 2.. Comparison of calculated and experimental membrane penetration depths of spin-labeled Cys residues in TM proteins. Five TM proteins were studied: MscL channel (1msl, open square), FepA receptor (1fep, black triangle), KcsA channel (1r3j, gray triangle), bacteriorhodopsin (1py6, black diamond), and BtuB porin (1nqe, black circle). The experimental distances are taken from the original publications (Altenbach et al. 1990, 1994; Greenhalgh et al. 1991; Klug et al. 1997; Perozo et al. 1998, 2001; Fanucci et al. 2002) but counted relative to the depth where parameter Φ is equal to zero. Points with zero depth correspond to residues that have been identified as first or last in the membrane-embedded segments of α-helices of β-strands. Calculated depths are defined as distances from Cβ-atoms of the corresponding residues to the closest boundary plane.
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Table Table 4.. Comparison of calculated and experimentally determined membrane-embedded portions of α-helices or β-strands
The calculated position of the retinal β-ionone ring in bovine rhodopsin along the bilayer normal (−4.1 Å) corresponds well to its location in 2D crystals (between sections z = 0 and z = −6 Å; Krebs et al. 2003). Four interfacial Trp residues of OmpA are located at distances of 8 Å to13 Å from the calculated bilayer center, close to the value of 9 Å to 10 Å obtained by the parallax method (Kleinschmidt and Tamm 1999). Several Trp residues of α-hemolysin (7ahl) are situated in the lipid head group area according to our results, which is consistent with their accessibility to water-soluble iodide and doxyl probes (Raja et al. 1999) and with locations of lipid head groups determined crystallographically (Galdiero and Gouaux 2004). The shallow location of Trp452 from the γ subunit of the nicotinic acetylcholine receptor (1.4 Å below the calculated hydrophobic boundary) is also consistent with fluorescence studies (Chattopadhyay and McNamee 1991).
Comparison with environments of residues
The water- or lipid-facing environments of individual residues in TM proteins can be mapped by different chemical probes. Lactose permease LacY (1pv6) has been more extensively studied by chemical modification than any other TM protein. A total of 393 single-Cys mutants of LacY were modified by bifunctional reagents to identify residues that could be involved in intermolecular cross-linking (Guan et al. 2002; Ermolova et al. 2003). Only residues located in regions of sufficiently high polarity could produce the reactive thiolate anion required for the formation of intermolecular disulfide bonds. Indeed, our calculations show that most of the residues susceptible to cross-linking are situated in water-exposed periplasmic and cytoplasmic loops or in a relatively narrow (∼5 Å) layer of the hydrophobic core where the dielectric permittivity may be intermediate between that in lipid and water (Fig. 3A). These layers are parallel to the calculated interfacial planes, thus confirming that these planes were identified correctly. However, some of the reactive residues (Trp78, Phe398) are situated 7–10 Å from the surface, which is most probably because they occupy polar sites at the C-termini of TM helices close to Lys74, Lys188, and Lys289 where local dielectric constant may be higher.
Figure Figure 3.. Comparison of calculated membrane core boundaries with experimental data for lactose permease (1pv6) in lipid bilayer (A,B), for rhodopsin (1gzm) in native membrane (C) and in detergent (D), and for OmpX (1qj8) in detergent (E). The boundaries are indicated by blue dots for the inner membrane side and red dots for the outer membrane side. (A) Cys-substituted residues in lactose permease, which were modified by cross-linking thiosulfonate agents (Cβ-atoms are colored red). (B) Residues of lactose permease that can be modified by NEM are colored red, residues inaccessible to NEM are colored blue, and lipid-accessible residues identified in spin-labeling studies are colored green. (C) Residues of rhodopsin that were modified by hydrophilic (red) and hydrophobic (blue) chemical probes in native photoreceptor membranes. (D) Spin-labeled residues of rhodopsin in detergent assigned to nonpolar and polar environments by EPR are colored blue and red, respectively (residues with undefined environment are colored gray). (E) NH and CH3 groups of OmpX that form NOEs with hydrophobic tails of DHPC are colored blue and green, respectively. NH groups that interact with head groups of DHPC are colored red. (F) A schematic representation of a protein in a native membrane (left) or in detergent (right).
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The calculated membrane boundaries of LacY are also consistent with site-directed modification of its 159 residues from TM helices II, VII, IX and X by N-ethylmaleimide (NEM) (Voss et al. 1997; Frillingos et al. 1998; Venkatesan et al. 2000a, b, c; Kwaw et al. 2001; Zhang et al. 2003). All residues inaccessible to NEM either face toward the lipid within the calculated hydrophobic slab, or are buried in the protein interior (blue in Fig. 3B). All residues modified by NEM are either accessible to water (outside the membrane boundaries, or in the large interior channel of the permease), or are situated at the water–lipid interface (for example, F308; red in Fig. 3B). Furthermore, site-directed spin-labeling studies of TM helices IV, V, and XII identified a number of residues that presumably face the lipid phase judging from their low accessibility to chromium and high accessibility to oxygen (Voss et al. 1996, Zhao et al. 1999). All these residues are located within the calculated boundaries and are exposed to lipid (green in Fig. 3B).
Vertebrate rhodopsins have also been studied in great detail. Environments of many rhodopsin residues were characterized in intact photoreceptor membranes (Davison and Findlay 1986a, b) or in n-dodecyl-β-D-maltoside (DM) (Hubbell et al. 2003). The results are consistent with the data for the native membranes, primarily with the results of chemical modification of ovine rhodopsin by hydrophobic and hydrophilic probes, which interact with residues exposed to nonpolar or polar environments, respectively (Barclay and Findlay 1984; Davison and Findlay 1986a, b). The hydrophobic probe was shown to modify all 14 Cys, Trp, Tyr, and His residues within the calculated hydrophobic boundaries (blue in Fig. 3C) and a few residues situated just outside the boundary in the lipid head group area (His65, Lys66, Tyr74, Lys231, Cys316) (Davison and Findlay 1986a). All polar residues that were modified by nonpermeable hydrophilic probe applied from the intracellular side (Barclay and Findlay 1984) are located outside the calculated hydrophobic slab (red in Fig. 3C).
The accessibilities to paramagnetic quenchers of two residues in Na+/H+ antiporter (1zcd) and eight residues in sensory rhodopsin II (1h2s) (Wegener et al. 2000; Hilger et al. 2005) are consistent with locations of the membrane boundaries calculated for the corresponding proteins.
Comparison with studies of TM proteins in detergents
The hydrophobic dimensions of TM proteins have also been studied in detergents using neutron diffraction with contrast variation in crystals (two photosynthetic reaction centers, trimeric porin OmpF and monomeric phosphoripase OmpLA), solution NMR (OmpX) and spin-labeling (rhodopsin). Detergent molecules form monolayers around nonpolar surfaces of TM proteins and most of them are oriented perpendicular to the protein surface, unlike lipids in bilayers (Fig. 3F; le Maire et al. 2000). In crystals of TM proteins, these monolayers look like regular rings with dimensions of 15–20 Å in the direction perpendicular to TM domains and 15–30 Å parallel to them (Roth et al. 1989, 1991). The latter values are in agreement with hydrophobic thicknesses of the corresponding proteins calculated by PPM 1.0 (Table 5). Moreover, the calculated membrane boundary planes of the proteins closely correspond to the borders of the detergent monolayer. For example, these planes pass through the aromatic rings of Trp78, Trp98, Phe109, and Phe122 residues in monomeric phospholipase A (OmpLA) in agreement with neutron diffraction (Snijder et al. 2003).
Table Table 5.. Comparison of calculated protein hydrophobic lengths (Dcalc) and experimental thicknesses of detergent monolayers around the proteins, as determined by neutron diffraction with contrast variation (Dexper)
A higher resolution picture of detergent–protein interactions has been obtained by solution NMR studies of OmpX β-barrel (1qj8) in the presence of a small amphiphile, 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) (Fernandez et al. 2002). Importantly, this NMR study identified environments of individual atoms in solution rather than in the crystal. A large set of aliphatic and NH hydrogens involved in NOEs with hydrophobic tails and head groups of DHPC has been determined (Fernandez et al. 2002), which allowed mapping of the detergent embedded area of the protein with high precision. The membrane boundaries calculated with PPM 1.0 are in agreement with NMR data (Fig. 3E). Only two NH backbone groups that interact with detergent occupy an “aromatic spot” outside the calculated slab.
Results of the calculations with “detergent” and “bilayer” scales (Table 1) were nearly identical for all proteins studied in detergents, except rhodopsin. To reproduce the experimental conditions, we used the crystal structure of rhodopsin with extended helix V (1gzm), removed its C-terminal palmitates, and applied the “detergent” solvation parameters. This led to an increased hydrophobic thickness (from 32.4 Å to 36.9 Å) and tilt angle (from 8° to 16°) (Fig. 3D). The expanded membrane boundaries are in much better agreement with spin-labeling data in DM (Hubbell et al. 2003) than with chemical modification data in native membranes (Davison and Findlay 1986a, b). The EPR studies identified a number of interfacial residues that were buried from water when substituted by a spin-labeled cysteine (V63, P71, V137, H152, K231, T251, and N310). The Cβ-atoms of these residues are indeed situated within the expanded boundaries calculated with “detergent” parameters, but well outside the boundaries obtained with “membrane” parameters (Supplemental Material, Table 3). Most importantly, several residues in the last turn of helix V (227–231) were shown to be coated with the detergent (Hubbell et al. 2003), but are accessible to water in the native membrane.
Application of the method to TM proteins from the PDB
After successful testing of the method for 24 well-studied TM proteins, it was applied to all other TM proteins deposited in the PDB. The calculations were conducted for 109 TM protein complexes (80 α-helical, 28 β-barrels, and gramicidin A dimer), 32 representative integral monotopic and peripheral proteins selected from the literature, and a control set of 20 water-soluble proteins with the highest hydrophobicity score in PDB_TM. Any protein that did not traverse the membrane after the optimization was interpreted as peripheral or monotopic, and the maximal membrane penetration depth of its atoms is calculated instead of its hydrophobic thickness.
Figure 4 shows that TM, integral monotopic and peripheral proteins occupy separate areas of the plot of hydrophobic thickness (D = 2z0) versus transfer energy (ΔGtransf), and therefore, they can be easily distinguished based on these two parameters. All peripheral and monotopic proteins have penetration depths of <15 Å. Sixteen hydrophobic water-soluble proteins from PDB_TM have transfer energies in the range from 0 to −0.5 kcal/mol. However, there are four exceptions that can be interpreted as probable membrane-associated proteins. Among them are VH antibody domain resistant to aggregation (1ohq, ΔGtransf = −7.2 kcal/mol), extracellular domain of bone morphogenetic protein receptor (1es7, ΔGtransf = −5.3 kcal/mol), gephyrin domain that links glycine receptor and tubulin (1t3e, ΔGtransf = −3.4 kcal/mol), and caseine kinase (1rqf, ΔGtransf = −1.6 kcal/mol). Indeed, interactions with membranes were shown to be functionally important for gephyrin and bone morphogenetic receptor (Sebald et al. 2004; Sola et al. 2004). Thus, our program can be applied for automatic identification and discrimination of TM, integral monotopic and water-soluble proteins, although the threshold between peripheral and nonmembrane proteins is sometimes blurred.
Figure Figure 4.. Hydrophobic thickness or membrane penetration depth (Dcalc) vs. transfer energy (ΔGtransf) plot for transmembrane (α-helical and β-barrel), integral monotopic, and peripheral proteins.
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Calculated hydrophobic thicknesses of TM proteins range from 21.1 Å to 43.8 Å, depending on the type of biological membrane (Table 6). Their average values are 29–30 Å for proteins from the inner bacterial, archaebacterial, endoplasmic reticulum and thylakoid membranes, but slightly higher (∼31 Å) for proteins from eukaryotic plasma membranes and slightly lower (∼27 Å) for proteins in inner mitochondrial membranes. However, thicknesses of proteins from outer membranes of Gram-negative bacteria (∼24 Å) and especially cell wall membranes of Gram-positive bacteria (43.8 Å) significantly deviate from 30 Å. This trend was noted previously and explained by the specific lipid compositions of the corresponding membranes (Faller et al. 2004; Tamm et al. 2004).
Table Table 6.. Calculated hydrophobic thicknesses of proteins from different biological membranes (Dmin, Dmax, Daver)
Calculated tilt angles (τ) of TM proteins vary from zero in all symmetric complexes to 1°–6° in the majority of monomeric and hetero-oligomeric structures. A few proteins have larger tilt angles, 7°–11°: rhodopsin (1gzm), mitochondrial succinate dehydrogenase (1zoy), SecY translocase (1rh5), and small monomeric β-barrels OmpA (1qjp), OmpX (1qj8), and NspA (1p4t). More extreme tilts (20°–26°) were obtained only for PagP enzyme from outer bacterial membrane (1thq), sulfohydrolase (1p49), different functional states of Ca2+-ATPase (1wpe, 1wpq, 1t5s, 1su4, 1iwo), the sensory domain of KvAP channel (1ors), and subunit c of F-type ATPase (1a91). A significant tilt of PagP was previously suggested based on the arrangement of its aromatic residues (Bishop 2005). Thus, TM proteins tend to be nearly perpendicular to the membrane, although the individual helices are tilted with respect to the bilayer normal by an average of 21° (Bowie 1997). All strongly tilted structures are either parts of incompletely assembled complexes (1gzm, 1zoy, 1rh5, 1ors, and 1a91), or have small TM domains and are therefore orientationally unstable (1qjp, 1qj8, 1p4t, and 1p49) or undergo large-scale conformational transitions (PagP and Ca2+-ATPase). Significant tilts are usually stabilized by peripheral helices that float in the membrane parallel to its surface, e.g., PagP enzyme, Ca2+-ATPase and rhodopsin.
D and τ parameters of TM proteins were prone to fluctuations within 1 kcal/mol around the global minimum of transfer energy. These fluctuations were usually smaller than 2 Å and 4°, respectively. However, the fluctuations were larger for proteins with a smaller TM perimeter (Fig. 5).
Figure Figure 5.. Dependence of fluctuations of hydrophobic thickness (A) and tilt angle (B) on the size of TM proteins. Transfer energy divided by hydrophobic thickness is roughly proportional to the length of the outer perimeter in TM proteins.
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Three proteins in our data set had unexpectedly small calculated hydrophobic thickness: EmrE transporter (1s7b), the first published structure of KvAP potassium channel (1orq), and heptameric mechanosensitive channel MscS (1mxm). This may reflect distorted, incomplete, or conformationally labile structures of these proteins. For example, according to our calculations, the EmrE dimer has very small hydrophobic thicknesses (16 Å), and an unusual tilt of TM (τ = 81°). On the other hand, the 3D structure of EmrE was described as inconsistent with cross-linking and biochemical data (Soskine et al. 2002, 2004; Butler et al. 2004) and with its EM image in 2D crystals (Ubarretxena-Belandia and Tate 2004). A small hydrophobic thickness (23 Å) of the KvAP tetramers (1orq) is probably related to the non-native arrangement of its three N-terminal helices in the crystal (Cuello et al. 2004; MacKinnon 2004; Long et al. 2005). Removing these three helices from the calculations resulted in a larger thickness (26 Å) and zero tilt angle, indicating that the rest of the structure is native. The relatively small (23 Å) calculated thickness of MscS mechanosensitive channel may be attributed to the open or another expanded state of the sensor, which is formed when the membrane becomes thinner under the influence of osmotic pressure (Bass et al. 2002; Akitake et al. 2005). Alternatively, this might be due to the disordered ends of TM helices, which do not include 25 N-terminal residues in each of the seven symmetric subunits.
In contrast, the hydrophobic thicknesses of F- and V-type ATPases and lipid flippases were unusually large, ∼36 Å (1a91, 1c17, 1yce, 2bl2, 1pf4, and 1z2r). This is expected to produce a significant hydrophobic mismatch between these proteins and their host bilayers. The mismatch may facilitate large-scale movements of these proteins in the lipid bilayers, because it reduces protein–lipid binding affinities (Lee 2003).
Some potential problems can also be detected based on the calculated tilt angles. These angles were close to zero for all TM complexes with noncrystallographic symmetry except cytochrome b6f from M. laminosus (1vf5) and fumarate reductase dimer from E. coli (1kf6), which both have τ of ∼3°. The non-zero overall tilt of cytochrome b6f complex appears due to significant differences between the symmetry-related subunits (RMSD of Cα-atoms ∼ 1 A). The dimer of fumarate reductase is loosely packed and therefore was suggested to be non-native (Iverson et al. 1999).
It is noteworthy that some TM proteins can form non-native dimers or trimers in crystals, for example, rhodopsin (1u19 and 1gzm), bacteriorhodopsin (1py6), lactose permease (1pv6), OmpA (1qjp), OmpX (1qj8), and fatty acid transporter FadL (1t16 and 1t1l). Hydrophobic thicknesses of such non-native oligomers are usually reduced (see Supplemental Material). Therefore, it is important to know the complete and correct quaternary structure of a multimeric complex to calculate its position in the membrane.