1Laboratory of Molecular Biophysics, The Rex Richards Building, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK and 3Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4 2Present address: Department of Chemistry, University of Rome La Sapienza, Piazzale Aldo Moro 5, I-00185 Rome, Italy
4 To whom correspondence should be addressed. E-mail: mark{at}biop.ox.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: bacteriorhodopsin/loop/membrane protein/molecular dynamics/polyalanine
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental studies have demonstrated a large degree of structural autonomy for individual helices, even in the absence of the loops that normally connect them. Native membrane protein structures were assembled from fragments that were separately refolded in vitro or synthesized in vivo. Apart from membrane proteins such as bacteriorhodopsin (Liao et al., 1983, 1984
; Luneberg et al., 1998
; Marti, 1998
), rhodopsin (Yu et al., 1995
; Ridge et al., 1996
), adrenergic receptor (Kobilka et al., 1988
) and muscarinic acetylcholine receptor (Schöneberg et al., 1995
), these studies also include proteins that form helical bundles, such as the voltage-gated potassium channel (Stühmer et al., 1989
), yeast
-factor transporter (Berkower and Michaelis, 1991
) and lactose permease (Zen et al., 1994
). It was found that helical fragments can fold individually and assemble spontaneously to form a functional protein, although the functionality was in some cases considerably reduced (Marti, 1998
). For bacteriorhodopsin in particular, it has been demonstrated that the N-terminal fragments AB, AC, AD and AE and also the C-terminal fragment CG are completely folded in lipid micelles (Luneberg et al., 1998
; Marti, 1998
). In contrast, the shorter fragments from the C-terminus (DG, EG and FG) and the centre of the protein sequence (CD, CE and DE) do not form
-helices of the expected lengths. Nevertheless, upon reconstitution in phospholipid micelles, all complementary pairs of fragments assemble in the presence of retinal to regenerate the characteristic bacteriorhodopsin chromophore with high efficiency. It has also been shown that the AB, BC, EF and FG loops are not required for the assembly process, although they help stabilize the structure (Kahn and Engelman, 1992
; Marti, 1998
). Some
-helical peptides such as alamethicin aggregate in the membrane to form pores without any inter-helical connecting loops (Sansom, 1993
; Cafiso, 1994
). Synthetic analogues of alamethicin in which individual helices were covalently linked were not impaired in their functionality, but showed primarily conductance levels corresponding to pores formed by even numbers of helices (Woolley et al., 1997
; Borisenko et al., 2000
).
Loops are usually the most flexible part of a membrane protein (Pautsch and Schulz, 1998; Arora et al., 2001
). Hence the questions arise of not only whether a protein model will be stable without connecting loops, but also how the sequence and conformation of the loop will affect its dynamics. The present project aimed to address this question by investigating the properties of a helixloophelix motif in a membrane environment. The F and G helices from bacteriorhodopsin, with the short extra-cellular loop that connects them, were chosen as the helixloophelix motif. Experimentally, helices F and G have been investigated in a lipid bilayer environment (a) in isolation (Hunt et al., 1997
), (b) in combination but without a connecting loop (Hunt et al., 1997
) and (c) a single peptide fragment with the loop (Luneberg et al., 1998
). In isolation, helix F was found in a peripheral membrane-bound conformation, whereas helix G appeared to form a ß-structure in the membrane. Bringing both peptides together in the same membrane by fusion of vesicles and reconstituting them together showed no alteration of their conformations compared with their conformations in isolation. The FG fragment displayed a significant loss of helicity, suggesting that the fragment is partially misfolded. Another study of the individual bacteriorhodopsin helices in dimethyl sulfoxide (DMSO) solution using NMR found helix F in its native
-helical conformation. The structure of helix G could not be determined since this peptide proved insoluble in DMSO (Katragadda et al., 2001
). This, together with the findings that both the isolated helices and the FG fragment had a higher helical content in SDS detergent than in lipid micelles (Hunt et al., 1997
; Luneberg et al., 1998
), indicates that the membrane-like environment is important for the structure of these bacteriorhodopsin fragments.
In this study, we investigated the structure and dynamics of a helix pair, taken from the bacteriorhodopsin structure, in explicit lipid environments and in a simplified membrane-mimetic octane slab. We focused on the effect of the connecting loop between helices F and G, by comparing simulations with and without this loop. For comparison, we also performed simulations of the same helixloophelix structure, but with all residues replaced by alanine residues. This combination of simulations allows us to investigate the relative importance of the loop, the sequence of the loop and the sequence of the transmembrane helices for determining the structure and dynamics of a pair of interacting transmembrane helices.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The segment of bacteriorhodopsin containing helices F and G (64 residues: Arg164 to Arg227) was taken from the 1cwq ground-state structure at 2.3 Å resolution (Sass et al., 2000). Figure 1 shows the fragment containing helix F (28 residues: Val167 to Glu194) and helix G (27 residues: Val199 to Arg225) connected by a four-residue loop (Gly195 to Ile198). The helixloophelix fragment was simulated in inserted transmembrane configuration in a range of different environments both with and without loop. All sequences were terminated with acetyl (Ace) and NH2 groups. The fragments contain 10 charged and six aromatic residues. The protonation states of the charged groups were chosen such that residues buried in the membrane are in their neutral states, while solvent-exposed residues are charged. This means that all ionizable residues are in their charged states except Asp212 and Lys216, which are buried in the membrane. Two chloride ions (Cl) were added to the solvent to balance the charge of the system. Each simulation was repeated with all residues mutated to alanine but retaining the initial conformation from the bacteriorhodopsin crystal structure. The fragments are Ace(Ala)64NH2 for the helixloophelix motif and Ace(Ala)31NH2 together with Ace(Ala)29NH2 for the individual helices.
|
Octane and POPC membranes have been described in detail elsewhere (Ulmschneider et al., 2004). The main advantage of octane is its short reorientation time. This provides an extremely flexible hydrophobic slab that represents a good mimic of the hydrocarbon tails of a biological bilayer membrane. At the same time it equilibrates much faster and allows better sampling of the protein. The system (box size 5 x 5 x 7.5 nm) was equilibrated for 1 ns at 300 K with harmonic restraints on the protein to retain its initial configuration. Two pre-equilibrated POPC bilayers were used, one consisting of 84 lipids (box size: 5.3 x 5.2 x 7.3 nm) and the other of 128 lipids (box size: 5.4 x 6.8 x 9.9 nm). A cavity with the shape of the fragment was created for each system using a method which minimizes the distortion of the bilayer (Faraldo-Gómez et al., 2002
). Lipid parameters were taken from Berger et al. (1997)
and combined with the GROMOS96 force field as reported previously (van Gunsteren et al., 1996
).
Simulation parameters
For the octane systems, a twin-range cut-off of 0.8/1.4 nm for both LennardJones and Coulomb interactions was used. For the lipid bilayer simulations, the Coulomb cut-off values were changed to 1.0/1.8 nm to take account of the charges on the lipid head-groups. The choice of electrostatics treatment may affect the structure of the bilayer, with somewhat different results for a twin-range cut-off, a reaction field treatment or a lattice method such as Particle Mesh Ewald (Anezo et al., 2003), but we do not think that this is critical for the current study. A dielectric constant of
r = 1 was used for all systems. Simulations were run with a 2 fs integration time step and neighbour lists were updated every 10 steps. Water, octane, POPC and protein were each coupled separately to a heat bath with a temperature of 300 K and time constant
T = 0.1 ps using a Berendsen thermostat (Berendsen et al., 1984
). For the octane systems the compressibility in x and y was set to zero (i.e. fixed box size in x and y) and the pressure in the z direction was kept at 1.0 bar using weak pressure coupling (Berendsen et al., 1984
) with coupling constant
P = 1.0 ps and compressibility
z = 4.6 x 105 bar1. In the POPC simulations, pressure coupling was used in x, y and z with
P = 1.0 ps and ki = 4.6 x105 bar1. Bond lengths were constrained with LINCS (Hess et al., 1997
). For all simulations, the GROMOS96 43a2 force field (van Gunsteren et al., 1996
) as implemented in GROMACS (version 2.0) (Berendsen et al., 1995
) and the SPC water model (Berendsen et al., 1981
) were used. Table I lists all simulations. FG WL represents helices F and G with the loop present, FG NL helices F and G without loop, Poly WL polyalanine with connecting loop and Poly NL polyalanine without loop. All simulations were between 6 and 10 ns long.
|
The systems were analysed structurally (hydrogen bond network, DSSP (Kabsch and Sander, 1983), root mean square (RMS) deviation and fluctuation) and dynamically (centre of mass distances, tilt, kink and crossing angles). Furthermore, the interactions between the helices and of the helices with their environment were investigated (aromatic ring orientations).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The root mean square deviation (RMSD) is an indicator of the structural stability of a protein in a given environment. Table I shows the time-averaged RMSD values of the simulations. The FG octane systems exhibit a substantial structural destabilization in the absence of the loop. However, this is in marked contrast to the behaviour of the POPC systems (WL 84, NL 84, WL 128 and NL 128), where the RMSD does not increase in the absence of the loop. Owing to a loss of helicity between residues Ser183 and Ile191, the simulation of the FG fragment with loop in a 128 POPC membrane has a much higher RMSD for helix F than its counterpart without loop. This behaviour was not observed in any of the other simulations. Corresponding values for the polyalanine simulations suggest that the loop does have a stabilizing effect in all systems, although it is only small. The reason why this effect is easier to detect in the polyalanine systems is perhaps the absence of other stabilizing groups. Aromatics and charged groups are abundant in the loop region (see Figure 1) and are thought to help stabilize transmembrane helices in the membrane (de Planque et al., 1999), while the many glycine residues in the loop are known to facilitate helixhelix packing (Eilers et al., 2000
; Senes et al., 2000
).
Table I shows that removal of the loop causes no discernible loss of local secondary structure on any of the systems investigated. Most helices remain -helical with the exception of the FG WL 128 system, where helix F shows substantial unfolding. Helix F has been shown to unfold partially in an octane membrane on its own (M.B.Ulmschneider, D.P.Tieleman and M.S.P.Sansom, unpublished data). However, it remained mainly helical in all other simulations. Local loss of helicity occurs chiefly at the helix termini and at residues known to act as pivots/hinges for kinking (see below). Interestingly, the polyalanine systems show an increased helicity in octane. This is due to the fluid-like behaviour of octane, which means that the extremely hydrophobic helices can tilt to bury most of their secondary structure in the octane slab, favouring an
-helical conformation. The POPC membrane is much more rigid, with the arrangement of the lipids allowing for only small tilt angles. Tilt analysis confirmed that the polyalanine helices are indeed more tilted in octane than in POPC (see below).
Analysis of crystal structures (Luecke et al., 1999) has revealed a set of inter-helical hydrogen bonds between helices F and G. In addition, the loop connecting helices F and G is stabilized by a number of backbone hydrogen bonds between mainly hydrophobic residues (see Figure 2). Although the polyalanine fragments retained all the backbone hydrogen bonds of the loop region in their starting conformation, they lost all inter-helical bonds except one. The starting structure of the polyalanine fragment has two additional backbone hydrogen bonds (A and B in Figure 3) that were not present in the FG fragment. These bonds are due to a more favourable backbone conformation after minimization and equilibration. A comparison between the time-averaged number of inter-helical hydrogen bonds is given in Table I. It was found that for the absence of a loop results in a greatly reduced number of hydrogen bonds for both octane systems (FG and polyalanine). Figure 3 shows a snapshot of the equilibrated starting conformation and another snapshot after 2 ns. All hydrogen bonds in the loop region were lost within the first nanosecond of the simulation owing to the helix termini moving apart. The remaining hydrogen bond between Thr170 and Ser226 was also lost after 3 ns.
|
|
It seems from the previous sections that the removal of the loop causes a destabilization of the helixloophelix motif, which is more pronounced in octane. The question arises of which regions of the fragments have become more flexible in the absence of a loop. Figure 4 shows the root mean square fluctuation (RMSF) with respect to the time-averaged structure for the 84 POPC (FG and polyalanine) simulations, which are representative of all the other simulations. In the FG systems, the loop region shows no increase in RMSF compared with the two helices, whereas the systems without loop show increased flexibility at the helix termini. This is in contrast to the polyalanine systems, where the loop manifests itself as a region of significantly increased RMSF, which is comparable in magnitude to the fluctuations at the helix termini in the simulations without loop. This suggests that the actual sequence of the loop might be more important for stabilization of a helixloophelix motif than the presence of a loop as such. Indeed, the FG loop is stabilized by four hydrogen bonds (hydrogen bonds 69 in Figure 2), whereas only one of these is present in the polyalanine loop. An interesting feature of the polyalanine simulations is the regular local increases in RMSF (see Figure 4). These are caused by residues on one helix that are in van der Waals contact with residues on the other helix. This behaviour is more pronounced in the WL system, indicating that the packing of the helices is slightly disrupted by the absence of the loop. This was confirmed by an analysis of the crossing angles (see below).
|
The ability of helices to kink has been associated with membrane protein function (Brandl and Deber, 1986; Deber et al., 1990
; Sansom and Weinstein, 2000
; Tieleman et al., 2001
). Indeed, it has been suggested that a kink of helix G found in a recent high-resolution crystal structure plays a role in the photocycle of bacteriorhodopsin (Luecke et al., 1999
). Furthermore, the proline-induced kink of helix F is believed to play a role in the opening of the cytoplasmic channel, providing solvent access to the retinal after isomerization (Subramaniam and Henderson, 2000
) and has previously been studied in some detail using molecular dynamics methods (Sankararamakrishnan and Vishveshwara, 1993
; Iyer and Vishveshwara, 1996
).
All simulations exhibited kinking for both helices F and G (see Figure 5). Two pivots were found in helix F: Val177Thr178 and Ser183Tyr185. Helix G has one hinge at Ser214Lys216. The -bulge found in the crystal structure near residues Ala215Lys216 was retained in the simulations. Whereas the magnitude of kinking varies between the different systems, the pivotal positions are identical in all simulations. Nevertheless, the POPC simulations restrict themselves to the Ser183Tyr185 hinge in helix F and also generally kink much less (helix F, 22 ± 7°; helix G, 23 ± 6°) than the octane systems (helix F, 40 ± 7°; helix G, 41 ± 18°). Generally, kinking differs between the NL and WL systems as seen in Figure 5, with helices F and G displaying average kink angle differences of 13 ± 6° and 10 ± 6° in the absence of the loop (values were averaged over all FG systems). The NL systems generally kink away from each other on the loop side, possibly as a result of the charged Glu194 and Glu204 residues. Figure 3 shows helix F kinking away from a straight helix G whereas in Figure 5 helix G kinks away from helix F.
|
The distance between the centres of mass of the individual helices was calculated in order to investigate how much the helices move with respect to each other. The results for the FG systems are shown in Figure 6A and B. In the absence of a loop, the centre of mass distance increases by several ångstroms, whereas the systems with loop retain their initial centre of mass distance of 10 Å. This effect is independent of the membrane environment. The polyalanine simulations exhibit a similar behaviour (Figure 6C and D). Interestingly, the octane simulation without loop approaches the value of the simulation with loop after fluctuating at a slightly higher value for 7.5 ns. In contrast, the simulation without loop in the 84 POPC membrane retains a larger separation than its counterpart with loop. The closer packing of the octane system was found to be due to an increased helix crossing angle in octane (see below). Even though all helices remain closely connected, the absence of the loop seems to increase the ability of the helices to move with respect to each other.
|
Tilt motions of the two helices are strongly correlated in the POPC systems (87% with loop and 77% without loop) and follow the total tilt of the helixloophelix motif. This is reflected in the crossing angles, which vary little during the 10 ns simulation time, with the FG WL 84 system having 15 ± 2° and the NL system 12 ± 2°. This suggests that the helixhelix motif is stable even in the absence of the loop, at least on the 10 ns time-scale of the simulations. Octane simulations, on the other hand, reveal significantly larger fluctuations of the crossing angles of about 7°. They too show no correlation of the helix tilt angles, regardless of whether a loop is present or not.
Polyalanine follows a similar pattern, with the octane systems exhibiting stronger tilt angles than the bilayer simulations. The crossing angle of the octane system with loop stabilizes within 200 ps at 17 ± 3°, while the simulation without the loop fluctuates for 8 ns before stabilizing at 19 ± 3°. This value is interesting since a crossing angle of 20° allows the ridges of one
-helix to fit into to grooves of another, providing for a closer packing of the motif (Chothia et al., 1977
) (see Figure 7). Indeed, it can be seen that the stabilization at 19° for the NL system is accompanied by a decreased distance between the helices (see Figure 6C). This also explains the peaks observed for the RMSF analysis in Figure 4, since residues in the grooves are restricted in their movements. The peaks are more pronounced for the WL system, which stabilizes much earlier near 20°. The crossing angles of the POPC systems were found to stabilize earlier in the simulations with both the 84 POPC WL and NL simulations stabilizing after 2 ns at 6 ± 2° (NL) and 8 ± 2° (WL). The 128 POPC simulations behave similar but with larger crossing angles of 13 ± 2° (NL) and 15 ± 1° (WL).
|
Since the earliest membrane protein structures, the abundance of aromatics at the interfacial region has been noticed (von Heijne, 1997). Aromatic properties include mechanical stability (Kreusch and Schulz, 1994
) and properties in connection with the membrane interface (Schiffer et al., 1992
; Hu and Cross, 1995
; Killian et al., 1996
). Some previous simulation studies have found aromatic anchoring at the membrane interfaces, in particular for tryptophan and tyrosine (Ulmschneider et al., 2004
). Others have found no evidence for conformational stabilization (Tieleman et al., 1998
). The analysis was restricted to the three residues near the membrane interface, namely Phe171, Trp189 and Phe208 (see Figure 1). Deletion of the loop had no effect on the accessibility of these aromatic residues.
Phe171 has no preferred orientation in any of the simulation systems. Trp189, on the other hand, displays a clear orientational preference for angles near 135° (see Figure 8), which is comparable to 150° from the crystal structure (1cwq.pdb), where
is the angle of the aromatic ring with respect to the membrane normal. Owing to the symmetry of the membrane, this is a good indicator of aromatic stabilization. The simulation with loop in octane remains at this angle for most of the simulation time, but after 8.2 ns it briefly flips by 90°, remaining at an angle of 45° with respect to the membrane normal for
120 ps before returning to its original orientation. In the corresponding simulation without loop, this flipping motion is much more prominent. Here the aromatic ring flips six times between the 135° and 45° states spending a roughly equal amount of time at each orientation. Time-scales of the flipping vary between 5 and 150 ps. This suggests that the presence of the loop adds to the stabilization of the tryptophan. No flipping is found in any of the lipid bilayer systems, which remain at a
angle of 135°.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the fundamental questions in computer simulations of biological systems is how well the equilibrium state has been sampled. For an adequate simulation of protein folding, unfolding or biological functions at atomic level the time-scales would have to be increased by several orders of magnitude, which is currently unfeasible for large-scale membrane protein simulations (Forrest and Sansom, 2000). However, analysis of structural stability is within the realm of current (i.e. multi-nanosecond) simulation times (Hansson et al., 2002
). Even though the FG helixloophelix motif represents a fairly small system, not all properties investigated have sampled their equilibrium equally well. Thermodynamic properties such as system volume, temperature and pressure are fully converged. While water and octane properties (density, orientation, diffusion coefficients) have explored the equilibrium well, POPC properties such as area per lipid require tens of nanoseconds to converge (Anezo et al., 2003
). The protein fragment is the most complex molecule in the system and many of the properties investigated converge on drastically different time-scales. The number of inter-helical hydrogen bonds can be regarded as satisfactorily sampled. Secondary structure and the motion of the aromatic rings show convergence and both the RMSF and RMSD flatten out at a certain value. However, this still does not guarantee their convergence. Among the longest time-scale motions are kinking and tilting and also translational motions of the helices in the membrane. These properties have to be understood as indicators of flexibility. Furthermore, there are differences between the types of membrane used. Simulations in octane generally show larger RMSD, RMSF and tilt and kink angles than the corresponding bilayer systems. This is due to the different reorientation times of octane and lipids. It should be noted the simulation time would have to be increased by orders of magnitude to determine fully the effect of a loop, which is not possible at present.
Role of the loop
The current set of simulations allows probing of the influence of a loop on the stability and dynamics of a helixhelix system in variety of different ways. First, by comparing the dynamics of the FG fragments in a POPC lipid bilayer, we can study the behaviour of a biologically realistic system in the presence or absence of a loop. Second, since the loop is located at the membranewater interface, which is a complex environment in a lipid bilayer, the simulations were repeated with an octane membrane, thus providing a simplified interface. Effects due to the loop being at the interface can therefore be separated from the effects of the loop as a means of connecting two helices. Third, a repetition of the above experiments with polyalanine fragments should allow one in principle to differentiate effects which are due to the loop per se and effects caused by the actual sequence of the loop.
The RMSDs and RMSFs give a good indicator of the complex interplay between sequence, presence or absence and environment of the loop. Results for the FG fragment show that the loop helps stabilize the helix termini to which it is connected, whereas for polyalanine this was not the case (see Figure 4). This suggests that the actual sequence and the packing are very important for the stabilizing properties of a loop. The four residues in the FG loop are stabilized by four hydrogen bonds, which are not present in the polyalanine loop. Consequently, the polyalanine loop is significantly more flexible. This effect was observed independently of the protein environment, again stressing the importance of the sequence.
The structural and hydrogen bond analysis indicates that the loop has stabilizing properties in all environments, albeit usually being of a weak nature. Only for the octane simulations of the FG fragment was a stronger stabilization due to the presence of the loop observed. Most of the important stabilizing hydrogen bonds are near the loop region. These are lost in octane owing to the increased number of water molecules compared with the POPC interface, explaining why the hydrogen bonding is the same in the absence of a loop for POPC but not for octane membranes. This suggests that the lipid bilayer interface compensates to a large extent for the absence of the loop, either directly through interactions of the loop termini with the lipid head-groups or indirectly by shielding the loop termini from the water and providing a constrained high-density environment that favours helixhelix interactions. Again, differences between the FG fragments and the polyalanine simulations highlight that the sequence of the loop termini is crucial for the effect caused by the polar lipid head-group environment, which is much weaker for polyalanine loops.
Most of the effects are visible only at the termini of the helices and do not necessarily reflect the stability of the fragment as a whole. Indeed, the crossing angles of the FG fragments vary only by a small amount between systems, suggesting that the helixhelix motif is conformationally stable even in the absence of the loop. Interfacial aromatics display a slight rotational stabilization as a consequence of the loop.
From the above analysis, it seems that the sequence and especially the packing of the loop are very important for structural stability at the loop side of the two helices. For POPC bilayer membranes the polar lipidwater interfaces substantially counteract the destabilization caused by a removal of the loop. However, this stabilization is still very much dependent on the sequence, being weaker for polyalanine. For octane membranes there is no polar interface and hence termini are slightly destabilized in the absence of a loop.
Conclusions
The current detailed comparative analysis indicates that the removal of the connecting loop has only a slightly destabilizing effect on a helixhelix pair embedded in a membrane. The amino acid sequence of the loop and adjacent to the loop appears to be more important than the presence of a loop as such. For a lipid bilayer membrane, our results suggest that the interfacial regions can compensate for inter-helical interactions lost upon deletion of the loop, at least for the time-scales investigated. The reason why destabilization is only detectable in octane rather than POPC could be due either to the slow reorientation times of lipids compared with octane or to the simplified nature of the octane slab membrane mimetic. However, even in the octane membrane the helix pair is stable and removal of the loop has only limited effects on the structure and dynamics of the helices. These effects are restricted to the helix termini. The sequence of the fragment at the interfaces is important for the magnitude of the effect; the destabilizing effect is much weaker for a neutral polyalanine helixloophelix motif than for the bacteriorhodopsin FG fragment. The current study is in line with the experimental observation that it is possible to reassemble functional membrane proteins from helical fragments.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arora,A., Abildgaard,F., Bushweller,J.H. and Tamm,L.K. (2001) Nat. Struct. Biol., 8, 334338.[CrossRef][ISI][Medline]
Berendsen,H.J.C., Postma,J.P.M., van Gunsteren,W.F., Hermans,J. (1981) Intermolecular Forces. Reidel, Dordrecht.
Berendsen,H.J.C., Postma,J.P.M., Vangunsteren,W.F., Dinola,A. and Haak,J.R. (1984) J. Chem. Phys., 81, 36843690.[CrossRef][ISI]
Berendsen,H.J.C., van der Spoel,D. and van Drunen,R. (1995) Comput. Phys. Commun., 95, 4356.
Berger,O., Edholm,O. and Jahnig,F. (1997) Biophys. J., 72, 20022013.[Abstract]
Berkower,C. and Michaelis,S. (1991) EMBO J., 10, 37773785.[Abstract]
Borisenko,V., Sansom,M.S. and Woolley,G.A. (2000) Biophys. J., 78, 133548.
Brandl,C.J. and Deber,C.M. (1986) Proc. Natl Acad. Sci. USA, 83, 91721.
Cafiso,D.S. (1994) Annu. Rev. Biophys. Biomol. Struct., 23, 141165.[ISI][Medline]
Chothia,C., Levitt,M. and Richardson,D. (1977) Proc. Natl Acad. Sci. USA, 74, 41304134.
Deber,C.M., Glibowicka,M. and Woolley,G.A. (1990) Biopolymers, 29, 14957.[CrossRef][ISI][Medline]
de Planque,M.R.R., Kruijtzer,J.A.W., Liskamp,R.M.J., Marsh,D., Greathouse,D.V., Koeppe,R.E., de Kruijff,B. and Killian,J.A. (1999) J. Biol. Chem., 274, 2083920846.
Eilers,M., Shekar,S.C., Shieh,T., Smith,S.O. and Fleming,P.J. (2000) Proc. Natl Acad. Sci. USA, 97, 57965801.
Faraldo-Gómez,J.D., Smith,G.R. and Sansom,M.S.P. (2002) Eur. Biophys. J., 31, 217227.[CrossRef][ISI][Medline]
Forrest,L.R. and Sansom,M.S.P. (2000) Curr. Opin. Struct. Biol., 10, 174181.[CrossRef][ISI][Medline]
Hansson,T., Oostenbrink,C. and van Gunsteren,W.F. (2002) Curr. Opin. Struct. Biol., 12, 190196.[CrossRef][ISI][Medline]
Hess,B., Bekker,J. Berendsen,H.J.C. and Fraaije,J.G.E.M. (1997) J. Comput. Chem., 18, 14631472.[CrossRef][ISI]
Hu,W. and Cross,T.A. (1995) Biochemistry, 34, 1414714155.[CrossRef][ISI][Medline]
Hunt,J.F., Earnest,T.N., Bousche,O., Kalghatgi,K., Reilly,K., Horvath,C., Rothschild,K.J. and Engelman,D.M. (1997) Biochemistry, 36, 1515615176.[CrossRef][ISI][Medline]
Iyer,L.K. and Vishveshwara,S. (1996) Biopolymers, 38, 401421.[CrossRef][ISI][Medline]
Jones,D.T. (1998) FEBS Lett., 423, 281285.[CrossRef][ISI][Medline]
Kabsch,W. and Sander,C. (1983) Biopolymers, 22, 25772637.[CrossRef][ISI][Medline]
Kahn,T.W., Engelman,D.M. (1992) Biochemistry, 31, 61446151.[CrossRef][ISI][Medline]
Katragadda,M., Alderfer,J.L. and Yeagle,P.L. (2001) Biophys. J., 81, 10291036.
Killian,J.A., Salemink,I., de Planque,M.R., Lindblom,G., Koeppe,R.E.,II and Greathouse,D.V. (1996) Biochemistry, 35, 10371045.[CrossRef][ISI][Medline]
Kobilka,B.K., Kobilka,T.S., Daniel,K., Regan,J.W., Caron,M.G. and Lefkowitz,R.J. (1988) Science, 240, 13101316.[ISI][Medline]
Kreusch,A. and Schulz,G.E. (1994) J. Mol. Biol., 243, 891905.[CrossRef][ISI][Medline]
Liao,M.J., London,E. and Khorana,H.G. (1983) J. Biol. Chem., 258, 99499955.
Liao,M.J., Huang,K.S. and Khorana,H.G. (1984) J. Biol. Chem., 259, 42004204.
Luecke,H., Schobert,B., Richter,H.T., Cartailler,J.P., Lanyi,J.K. (1999) J. Mol. Biol., 291, 899911.[CrossRef][ISI][Medline]
Luneberg,J., Widmann,M., Dathe,M. and Marti,T. (1998) J. Biol. Chem., 273, 2882228830.
Marti,T. (1998) J. Biol. Chem., 273, 93129322.
Pautsch,A. and Schulz,G.E. (1998) Nat. Struct. Biol., 5, 10137.[CrossRef][ISI][Medline]
Ridge,K.D., Lee,S.S. and Abdulaev,N.G. (1996) J. Biol. Chem., 271, 78607867.
Sankararamakrishnan,R. and Vishveshwara,S. (1993) Proteins: Struct. Funct. Genet., 15, 2641.[CrossRef][ISI][Medline]
Sansom,M.S.P. (1993) Q. Rev. Biophys., 26, 365421.[ISI][Medline]
Sansom,M.S.P. and Weinstein,H. (2000) Trends Pharmacol. Sci.., 21, 445451.[CrossRef][ISI][Medline]
Sass,H.J., Buldt,G., Gessenich,R., Hehn,D., Neff,D., Schlesinger,R., Berendzen,J. and Ormos,P. (2000) Nature, 406, 649653.[CrossRef][ISI][Medline]
Schiffer,M., Chang,C.H. and Stevens,F.J. (1992) Protein Eng., 5, 213214.[ISI][Medline]
Schöneberg,T., Liu,J. and Wess,J. (1995) J. Biol. Chem., 270, 1800018006.
Senes,A., Gerstein,M. and Engelman,D.M. (2000) J. Mol. Biol., 296, 921936.[CrossRef][ISI][Medline]
Stühmer,W., Conti,F., Suzuki,H., Wang,X.D., Noda,M., Yahagi,N., Kubo,H. and Numa,S. (1989) Nature, 339, 597603.[CrossRef][ISI][Medline]
Subramaniam,S. and Henderson,R. (2000) Nature, 406, 653657.[CrossRef][ISI][Medline]
Tieleman,D.P., Forrest,L.R., Sansom,M.S.P. and Berendsen,H.J.C. (1998) Biochemistry, 37, 1755417561.[CrossRef][ISI][Medline]
Tieleman,D.P., Shrivastava,I.H., Ulmschneider,M.B. and Sansom,M.S.P. (2001) Proteins: Struct. Funct. Genet., 44, 6372.[CrossRef][ISI][Medline]
Ulmschneider,M.B. and Sansom,M.S.P. (2001) Biochim. Biophys. Acta Biomembr., 1512, 114.[ISI][Medline]
Ulmschneider,M.B., Tieleman,D.P. and Sansom,M.S.P. (2004) J. Phys. Chem. B, 108, 1014910159.[ISI]
van Gunsteren,W.F., Krüger,P., Billeter,S.R., Mark,A.E., Eising,A.A., Scott,W.R.P., Hüneberger,P.H. and Tironi,I.G. (1996) Biomolecular Simulation: The GROMOS96 Manual and User Guide. Biomos/Hochschulverlag an der ETH Zürich, Groningen/Zürich.
von Heijne,G. (1997) Prog. Biophys. Mol. Biol., 66, 113139.[ISI]
Wallin,E. and von Heijne,G. (1998) Protein Sci., 7, 10291038.
Woolley,G.A., Biggin,P.C., Schultz,A., Lien,L., Jaikaran,D.C., Breed,J., Crowhurst,K. and Sansom,M.S. (1997) Biophys. J., 73, 770778.[Abstract]
Yu,H., Kono,M., McKee,T.D. and Oprian,D.D. (1995) Biochemistry, 34, 1496314969.[CrossRef][ISI][Medline]
Zen,K.H., McKenna,E., Bibi,E., Hardy,D. and Kaback,H.R. (1994) Biochemistry, 33, 81988206.[CrossRef][ISI][Medline]
Received April 29, 2005; accepted August 11, 2005.
Edited by Klaus Schulten
|