Comparison of the Adsorbed Conformation of Barley Lipid Transfer

Biomacromolecules 2008, 9, 1443–1453

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Comparison of the Adsorbed Conformation of Barley Lipid Transfer Protein at the Decane-Water and Vacuum-Water Interface: A Molecular Dynamics Simulation S. R. Euston,*,†,‡ P. Hughes,†,‡ Md. A. Naser,†,‡ and R. E. Westacott§ School of Life Sciences, International Centre for Brewing and Distilling, and School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 4AS, United Kingdom Received November 7, 2007; Revised Manuscript Received February 21, 2008

Molecular dynamics simulation is used to model the adsorption of the barley lipid transfer protein (LTP) at the decane-water and vacuum-water interfaces. Adsorption at both surfaces is driven by displacement of water molecules from the interfacial region. LTP adsorbed at the decane surface exhibits significant changes in its tertiary structure, and penetrates a considerable distance into the decane phase. At the vacuum-water interface LTP shows small conformational changes away from its native structure and does not penetrate into the vacuum space. Modification of the conformational stability of LTP by reduction of its four disulphide bonds leads to an increase in conformational entropy of the molecules, which reduces the driving force for adsorption. Evidence for changes in the secondary structure are also observed for native LTP at the decane-water interface and reduced LTP at the vacuum-water interface. In particular, intermittent formation of short (six-residue) regions of β-sheet is found in these two systems. Formation of interfacial β-sheet in adsorbed proteins has been observed experimentally, notably in the globular milk protein β-lactoglobulin and lysozyme.

Introduction The adsorption of proteins at the interface between two immiscible phases is a common occurrence in biological systems.1 This is due to the amphiphilic nature of proteins, which contain a range of amino acids with both hydrophobic and hydrophilic side chains. Therefore, there is a tendency for the hydrophilic regions of the protein to partition toward the aqueous side of an interface, while the hydrophobic regions partition to the hydrophobic (or nonaqueous phase). The adsorption of proteins can either be beneficial or detrimental depending on the type of protein and the biological system in question. In the biomedical sciences, preventing adsorption of proteins or removing adsorbed proteins from surgical instruments has become an important issue, particularly with the advent of prion diseases.2 On the other hand, the adsorption of proteins to bioimplants will decrease the likelihood of rejection by the body.3 An important area of research in biotechnology is the field of biosensors.4 In some cases, proteins are adsorbed onto inorganic substrates for use as sensors (e.g., an enzyme that has a particular analyte as a substrate). In the food and beverage industry, proteins are exploited as functional ingredients that adsorb to the surface of oil droplets and air bubbles to stabilize them against separation. Examples of these are milktype emulsions (oil droplets in water), whipped desserts, and the head (foam) on beer. All of these examples share a common feature, that is, a protein or proteins that will adsorb to the interface between the oil and water (for emulsions) or air and water (for foams), and they stabilize it against separation of the dispersed phase. There have been many studies on the properties of proteins in emulsions and foams.5,6 These have investigated the protein * To whom correspondence should be addressed. Tel.: +44 131 451 3640. Fax: +44 131 451 3009. E-mail: [email protected]. † School of Life Sciences. ‡ International Centre for Brewing and Distilling. § School of Engineering and Physical Sciences.

structure and their ability to stabilize interfaces, the kinetics of adsorption, and the gross structure of the adsorbed layer. Some studies have also attempted to look at the structure of the adsorbed protein in more detail, although this is not a trivial task. Most of these studies have looked at the changes in secondary structure that occur on adsorption.7–10 While this gives some information on the adsorbed protein conformation, it does not allow the elucidation of the full 3-D (tertiary) structure of the adsorbed protein. In fact, very little is known about the conformation that a protein adopts at an interface and, until recently, what information there was had been inferred from indirect experimental studies such as surface pressure–area isotherms.11 A major reason for this is that it has proven difficult to apply to adsorbed proteins those techniques used successfully to elucidate protein tertiary conformation in solution. Adsorbed proteins cannot be crystallized and, therefore, cannot be investigated with X-ray crystallography. High resolution NMR studies of adsorbed proteins have only been reported recently12,13 and have not as yet been applied to fluid interfaces relevant to foods and beverages. Theoretical and computational studies of protein adsorption have also been attempted. Most studies have involved the use of simplified, mesoscopic models for protein adsorption that treat disordered proteins as linear chains of rigid spheres14 and globular proteins as deformable particles.14–16 These models are good at representing the gross structural properties of adsorbed protein but at the expense of conformational detail. An alternative approach is to use self-consistent field methods, such as Scheutjens-Fleer theory,17 which have been extended to account for polyelectrolytes. While useful for linear polymers such as the caseins,17 this approach cannot account for the greater structural complexity of globular proteins. The development of simulation methods for protein adsorption has been hindered by the structural complexity of even the most simple protein molecules. However, progress has been made. Different

10.1021/bm701227g CCC: $40.75  2008 American Chemical Society Published on Web 04/18/2008

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Figure 1. NMR structure of native barley LTP, as determined by Heinemann et al. (ref 15). The detail of the atom positions has been removed from this conformation, and only the global fold (with helices in schematic form) and the four disulphide bonds are shown. In the figure, the N- and C-terminal ends of the molecule, the A, B, C, and D helices, loops 1, 2, 3, and the extended loop, and the disulphide bonds are indicated.

approaches have been taken to simulate disordered and globular proteins.14–23 Our own research has concentrated on the application of Monte Carlo simulation to study adsorbed protein conformation. We have used this approach to carry out the first simulated determination of the equation of state of adsorbed proteins.22 Another method that has been applied to small adsorbed proteins and that promises to be able to probe adsorbed protein conformation at the atomic scale is molecular dynamics (MD) computer simulation. There have been several studies of the adsorption of peptide fragments or whole proteins using MD simulation, and these have been summarized by Euston.23 To date, these studies have looked at either solid or lipid surfaces and/or small proteins that are chosen for their size and ease of computation rather than for their importance in biological or food systems. In this study, we have chosen to simulate barley lipid transport protein (LTP) adsorbing at an air–water interface. This protein is one of the major contributors to the stability of beer foam and, thus, it is an economically important protein.24,25 Barley LTP is one of two major protein contributors to beer foam stability,24,25 the other being protein Z, a member of the serpin family. The structure of LTP is shown in Figure 1.26 LTP is comprised of 91 amino acids (9.7 kDa molecular mass), with a secondary structure characterized by four R-helices connected by loop structures (Figure 1). The structure is stabilized by four disulfide bonds (Figure 1). The C terminal peptides Asn74–Tyr91 is in extended configuration with no obvious secondary structure.26 Heinemann et al.26 have labeled these helices with the letters A-D (Figure 1). Helices A-C form an up–down-up motif, with helix A in a central position and helices B and C on either side. The extended loop structure between residues Val75-Ile90 forms hydrophobic contacts with all fours helices. This structure is strengthened by the presence of the Cys48-Cys87 disulphide bond.26 LTP also has a hydrophobic cavity that is the binding site for hydrophobic and amphipilic lipids.27 This is formed from hydrophobic residues at the inner face of the four helical regions.27 These structural factors contribute to the high conformational stability of LTP in solution, and it is

Euston et al.

reported to have a denaturation temperature around 100 °C.28 In its native form, LTP does have some foam forming and stabilizing ability. However, during the wort boiling stage of the brewing process (wort is a clarified extract, principally of malted barley, that is typically boiled for 1–2 h during commercial beer production to develop flavor, stabilize the extract, and precipitate polyphenols), LTP has been found to be denatured (at least partially), become reduced (one or more disulphide bonds are broken), and to undergo varying degrees of glycosylation.29 The resulting LTP molecules have a higher foaming ability.29 The stability of beer foam is complex and controlled not only by the protein components of the system, but also by other small molecule components. The hop-derived iso-R-acids are a particularly important group of compounds that contribute to foam formation and stability. Hughes and Wilde30 have shown that these contribute to foaming by interacting with the adsorbed protein layer at the air–water interface and improving the rheological properties of the foam surface. The hydrophobic nature of the iso-R-acids also contributes to their bitter taste, and they are the major bittering component in beer.31 Our understanding of adsorbed protein conformation has advanced considerably over the past decade. However, this understanding is far from complete. Computer simulation has the potential to complement both experimental and other theoretical methods and to close some of the gaps in our knowledge. The ultimate goal of simulation methods would be to carry out a full atomistic simulation of the conformational changes that occur in an adsorbing protein. To date, however, few atomistic simulations of protein adsorption have been attempted.23,32,33 The main reasons for this is that MD simulations of large proteins are time-consuming and, at present, limited to submicrosecond timescales. In this paper we present results for a study of the adsorption of LTP at a vacuum-water and decane-water interface. The adsorption of the native LTP structure and a structure where all four disulphide bonds have been reduced has been modeled. Simulation Methodology. Molecular dynamics simulations were carried out using the Gromacs MD package34 version 3.3.1. Four systems were studied, (1) native barley LTP adsorbing at a water-vacuum interface; (2) reduced barley LTP adsorbing at a water-vacuum interface; (3) native LTP adsorbing at a decane-water interface; and (4) reduced LTP adsorbing at a decane-water interface. The starting conformation for all studies was the LTP structure determined by Heinemann et al.26 and deposited in the protein database as 1LIP. For the native LTP simulation, this structure was used unmodified. For the reduced LTP, the four disulphide bonds in the structure were cleaved by manual editing of the pdb file and the resulting structure used to initiate MD simulations. Setting Up the Simulation Box. A water box of size 6 × 6 × 6 nm was set up using the SPC water model. Periodic boundary conditions were defined in the three coordinate directions, and the water density was scaled to 1000 g/L by varying the number of water molecules. A water-vacuum interface was formed by expanding the simulation box in the z-direction to create two vacuum spaces at each z-face of the water box. Periodic boundary conditions were maintained on all sides of the vacuum spaces. The water box was then subjected to a 2 ns MD run to equilibrate the system. The initial simulation of the expanded water box in the absence of protein has shown that a stable water-vacuum interface is formed. Water molecules do detach from the water phase at the interface

Molecular Dynamics Simulation of LTP Adsorption

at regular intervals and move into the vacuum space. However, these do not remain in the vacuum space to form a vapor phase. The protein was inserted into the center of the water phase of the box after this equilibration period. The final number of water molecules for the two systems were 7440 for the native LTP system and 6959 for the reduced LTP system. Counter ions were added to neutralize the system. For the decane-water interface systems, a simulation cell was set up by defining a water box of dimension 6.5 × 6.5 × 5 nm filled with SPC water. Periodic boundary conditions were defined in the three coordinate dimensions. Two decane-water interfaces were formed by expanding the simulation cell by 2 nm at the two z-faces of the cubic water box and filling these spaces with decane molecules. Periodic boundary conditions were maintained at the x and y faces. Periodic boundary conditions were also maintained in the z-direction so that decane molecules moving across this boundary would rejoin the simulation cell in the decane layer on the opposite side of the box. The numbers of decane and water molecules were adjusted so as to scale the density of water and decane to 1000 g/L and 720 g/L, respectively. The SPC water model was used in all of the simulations. The dimensions of the decane layer (2 nm on each side, that is, a total thickness of 4 nm) was just under twice the diameter of the native LTP molecule in our simulations (approximate radius of gyration 1.2 nm). The decane/water cell was subjected to a 2 ns MD simulation run to equilibrate the box. A single molecule of LTP was then introduced in to the center of the water phase of the simulation cell. This required deletion of some of the water molecules in the box. The final number of water molecules in the simulation cell was 4923 for the native LTP system and 4845 for the reduced LTP system and the number of decane molecules was 408 for the native LTP and 409 for the reduced LTP systems. Counter ions were added to neutralize the system. MD Simulation Run. During the MD run, the interactions between various groups in the molecules in the system were modeled using the GROMOS96 force field35 and the SPC water model.36 The long-range electrostatic interactions were calculated using the particle mesh Ewald summation method (PME). A switching function was used to smoothly decay the Lennard-Jones interactions to zero after a certain cutoff distance. The cutoff was chosen as 0.9 nm, with the interactions decaying exponentially to zero at 1.2 nm. The temperature in the system was maintained at 300 K by coupling the whole system to a Berendsen thermostat37 using a coupling constant of 0.1 ps. Simulations were run for up to 40 ns, with a 2 fs time step, using the leapfrog algorithm as the integrator. Center of mass motion was removed from all atoms over the course of the simulation. In addition to the simulations noted above we also simulated native LTP adsorbing at the water-vacuum interface using a different force field, the OPLS all atom force field.38 Qualitatively, the results from this simulation were very similar to those from the simulation using the GROMOS force field. We interpreted this to mean that the results from the adsorption simulations were relatively independent of force field type and that the similarity of the two sets of results from different initial random velocities in the starting conformation meant that our results are a good estimate of the global behavior of the systems.

Analysis of Results The structures of the simulated adsorbed proteins were characterized in a number of ways. Changes in the secondary


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structure were analyzed by determining the number of amino acid residues existing in helices, β-sheet or in random coils as a function of simulation time using Deep View/Swiss-PdbViewer.39 Changes in the gross structure (tertiary structure) were estimated by following the root-mean-square deviation (RMSD) of the positions of all atoms in the structure from the starting conformation as a function of time using eq 1

RMSD(t1, t2) )

N

1⁄2

[ M1 Σ m |r (t ) - r (t )| ] 2

i

i)1

i 1

i 2

(1)

where ri are the vector positions of atoms at different times, N is the total number of atoms in conformation t1 and conformation t2, mi is a weighting factor usually equal to the mass of each atom, and M is the sum of all mi. The calculation of RMSD was carried out by comparing atom positions at time steps in the trajectory (t2), with their positions in a reference conformation (t1), which for our simulations was the t ) 0 time step. The radius of gyration of the LTP molecule was also used as an indicator of structural change and was calculated using eq 2

Rg )

[

1 N Σ w (r - rcm)2 N i)1 i i N

N Σ wi i)1

]

1⁄2

(2)

where rcm is the coordinates of the center of mass of the molecule and ri are the coordinates of each of the N atoms in the molecule. Density profiles of water, decane, and protein across the simulation cell were calculated. The change in the number of hydrogen bonds (protein–protein, protein-water, and water– water) was also calculated. Absolute configurational entropy of the protein molecule was calculated using Schlitter’s quasiharmonic approximation method.40 In this method, we estimate an effective Hessian matrix by calculating the covariance matrix of the internal coordinate variations from the MD trajectory and then use Schlitter’s equation40 to calculate the entropy from the eigenvalues of the covariance matrix. Finally, the number, type, and sequence position of amino acids in the interfacial region were determined.

Results and Discussion Native LTP at the Vacuum-Water Interface. Figure 2a shows a snapshot conformation at 23 ns of a native LTP molecule adsorbing at the water-vacuum interface. The details of the positions of all atoms in the amino acids has been removed, and only the global fold of LTP is shown. We find that in the first 5 ns of the simulation the protein does not adsorb to the surface but undergoes both translational and rotational movement away from its starting conformation. Around 5 ns, the molecule makes contact with the interfacial region and adsorbs to the water-vacuum interface. The molecule stays adsorbed to the interface for the rest of the simulation run up to 23 ns. There is little evidence for adsorption-induced unfolding of LTP from the snapshot conformation in Figure 2a. Figure 3 is a plot of the number of amino acid residues in helical and random coil regions as a function of simulation time. For the adsorbed native LTP structure, there is little if any change in the proportions of helix and coil structure throughout the simulation run. If we look at a plot of RMSD as a function of simulated time (in ns; Figure 4), we notice three things. First, there is a rapid jump in RMSD at the start of the simulation as the NMR structure used becomes solvated and equilibrated with the SPC water model. Second, we note that the RMSD slowly

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Figure 3. Secondary structural data for adsorbed native and reduced LTP molecules. The number of amino acid residues found in helical, β-sheet, or random coil regions is plotted as a function of simulation time. b ) native LTP helix; 1 ) native LTP sheet; 9 ) native LTP coil; O ) reduced LTP helix; 3 ) reduced LTP sheet; and 0 ) reduced LTP coil. (a). LTP adsorbed at the vacuum-water interface. (b). LTP adsorbed at the decane-water interface.

Figure 2. Snapshot conformation of (a) native LTP and (b) reduced LTP adsorbing at a water-vacuum interface after 23 ns (native LTP) and 32 ns (reduced LTP) of a MD simulation. The global fold of the LTP is shown as a solid ribbon. Only adsorbed amino acid residue side chains are shown. Amino acid residues that are adsorbed in the interfacial region are highlighted in black.

increases throughout the 23 ns of the simulation, which suggests a slow conformational change. Finally, we can see that the RMSD for the adsorbed native LTP is lower than for the native LTP in a water box, suggesting some conformational change occurs on adsorption. We can further analyze the conformation of the adsorbed LTP by following changes in the radius of gyration (Rg; Figure 5). The overall Rg remains constant throughout the simulation run, although again, there is a difference between the adsorbed native LTP and the native LTP in a water box (Rg is lower for the latter). If we analyze the components of Rg in the x, y, and z coordinate directions (x and y being parallel, and z being normal to the interface), however, we see some differences in the LTP conformation. After 5 ns, when the LTP molecule contacts the surface, there is a rapid jump in Rgy accompanied by a decrease in Rgz (data not shown). This suggests that upon adsorption to the interface the LTP molecule starts to flatten onto the interface, while elongating parallel to the interface, that is, there is a small degree of spreading. This is consistent with the changes observed in RMSD. The constant total Rg during the simulation (Figure 5) implies that this change in shape of the molecule occurs at a constant molecular volume. We have carried out a detailed analysis of the amino acid residues that are adsorbed to the

Figure 4. RMSD for adsorbed native and reduced LTP at both water-vacuum and decane-water interfaces as a function of simulation time. – ) native LTP at the water-vacuum interface; - - - ) reduced LTP at the water-vacuum interface; · · · · ) native LTP at the decane water interface; and - · · - · · - ) reduced LTP at the decane-water interface. The RMSD for native LTP in a water box (no interface) is included for comparison – – –.

interfacial region (Figure 2a and Figure 6). The interfacial layer is defined as the region of reduced water density at the edge of the water box. This region extends for approximately 1 nm at the vacuum-water interface. The interfacial region can be seen clearly in Figure 7, which shows the density profile for water (and protein) across the simulation box. The amino acid residues of the adsorbed native LTP that are in the interfacial region are shown in Figure 6, are marked in the snapshot conformation of Figure 2a, and colored black to distinguish them from nonadsorbed residues. Also marked in Figure 6 are the amino acid residues that define the hydrophobic binding pocket of LTP. Two main regions of the LTP molecule are involved in the initial adsorption to the interface. From Figure 6, we can see that these


Figure 5. Radius of gyration (Rg) for native and reduced LTP adsorbed at the decane-water interface and vacuum-water interfaces. The four lines in the plot correspond to s native LTP at the water-vacuum interface; · · · reduced LTP adsorbed at the vacuum-water interface; --- native LTP adsorbed at the decane-water interface; and – · · – · · – · · reduced LTP adsorbed at the decane-water interface. The RMSD for native LTP in a water box (no interface) is included for comparison – – –.

are from Ser8 to Asn29 and from Gly57 to Asn62. The majority of the adsorbed residues are found in the A and B helices and loops 1 and 3 (Figure 2a and Figure 6). During the course of the simulation, up to 23 ns, the amino acid residues in the interfacial region do not change a great deal. In general, these same two regions are adsorbed to the surface throughout, although there is some interchange between adjacent residues as the simulation progresses. Over the 23 ns of the simulation between 24 and 29 amino acid residues can be found in the interfacial region. Of these, 4–7 contain hydrophobic, 7–10 contain hydrophilic, and the remainder contain neutral side chains. The hydrophobic binding pocket of LTP contains 14 amino acid residues,27 which are also indicated in Figure 6. Of these, a maximum of only four are found in the interfacial region during the simulation. Comparison of the adsorbed residues with the native structure in Figure 1 reveals that these residues are at or close to the surface of the native molecule. This suggests that if there is conformational spreading at the interface it is not extensive. The driving force for adsorption in this system is the displacement of water from the interfacial region back into the bulk water. From the density profile for the native LTP/ water-vacuum interface system (Figure 7) we can see that the interfacial region is characterized by a region of decreasing water density. In this region, the water molecules have a reduced capacity to hydrogen bond because, on average, they are further apart. When the protein moves into the interfacial region, water is displaced into the bulk phase where its ability to hydrogen bond with other water molecules is increased. This can be seen in Figure 7 as a lower water density profile in the region where the protein is present. This results in a favorable net change in the free energy of the system, which in turn drives the adsorption process. Analysis of the water–protein and water–water H-bonds in the system shows no significant change upon adsorption of the LTP molecule. This suggests that the changes in H-bonds are small compared to the overall numbers in the system. Similarly, the number of protein–protein H-bonds does not change significantly, again suggesting no conformational changes upon adsorption. A further interesting feature of the density profile in Figure 7 is that the protein stays fully in the water phase and at no time does it penetrate significantly into the vacuum region. This suggests that the interaction between water and hydrophilic protein residues is sufficient to keep the protein in the water phase and acts to reduce the spreading of the protein


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at the interface. The density profile for the adsorbed LTP, where the protein density is at a maximum a short distance from the interface, is similar to those generated using lattice models for weakly adsorbing lattice chains or globules.22 Lattice chain simulations have shown that for weakly adsorbing chains the effect of conformational entropy acts to create a depletion region close to the adsorbing surface. Only when the adsorption interaction exceeds a certain critical value22 does the protein density become a maximum at the surface. For linear lattice chains this critical adsorption energy is about 0.5 kBT per adsorbing segment, while for adsorbing globules this value is lower.22 While a direct comparison of the adsorption energy per segment cannot be made with our MD simulations, this would suggest that the interaction between LTP and the airvacuum interface is relatively weak, which is consistent with the displacement of a relatively small number of water molecules from the interfacial region, and the consequent formation of a small number of new H-bonds in the bulk. In our relatively short 23 ns MD simulation, the LTP molecule adsorbs to the surface and remains in its native state. The small extent of surface-induced unfolding in LTP is not surprising for two reasons. First, the time scale of the simulation is short and it is known that spreading of proteins at interfaces occurs over time-scales far longer than ns.1 For some proteins, structural changes may even continue over time-scales of hours and days. One way to speed up the conformational change would be to carry out the simulation using an implicit water model with the appropriate dielectric constant, because explicit solvent increases the relaxation times of the protein molecule. Obviously, this approach would not explicitly account for the hydrogen bonds in the system, which might in turn affect the adsorption behavior of the protein. The second reason is that LTP is known to be a remarkably stable molecule that can withstand temperatures up to 100 °C before it denatures.27 This suggests that the free energy barrier to unfolding is large, and the driving force for unfolding that comes from molecular adsorption may simply not be enough for the native LTP molecule to unfold at the vacuum-water interface. In addition to this, there is still some doubt as to whether surface adsorption actually does lead to unfolding of secondary structure. Li-Chan et al.41 have used Raman spectroscopy to study adsorption-induced secondary structure changes in the conformation of bovine serum albumin (BSA) at an oil–water interface. They noticed no unfolding of the secondary structure (principally β-sheet in BSA) upon adsorption. Reduced LTP at the Vacuum-Water Interface. In beer foam it is unlikely that the LTP molecules still exist in their native state. During so-called wort boiling, the protein complement is subject to vigorous boiling, typically for 60–90 min. This treatment may result in the structure being reduced, denatured, and glycosylated to varying degrees.29 Partially denatured proteins are known to have altered adsorption properties and can exhibit enhanced ability to stabilize interfaces.42 We have also simulated the adsorption of an LTP molecule where all of the disulphide bonds have been reduced to determine whether this might lead to enhanced spreading of the protein at the interface. Figure 2b is a snapshot conformation of reduced LTP at a vacuum-water interface after 32 ns. The structure of the modified molecule is more flexible than the native conformation and the molecule has an altered conformation. We find that in this system adsorption of the protein at the interface has occurred by 5 ns of simulation time. What is noticeable is that the molecule does not appear to be as strongly attached to the interface, as does the native LTP structure (compare Figure 2b with Figure 2a). This may be an entropy

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Figure 6. Amino acids present in the interfacial region at the end of the simulation of native and reduced LTP molecule adsorbing at the vacuum-water and decane-water interfaces. The presence of an amino acid in the interfacial region is indicated by a coloured block: black ) hydrophobic residue; dark gray ) hydrophilic residue; light gray ) neutral residue. The amino acids found in the binding site of LTP are also indicated in the diagram. The assignment of the individual amino acids to the structural features of LTP (helix or loops) is also indicated.

effect. By reducing the LTP molecule, we increase its flexibility and therefore increase its conformational entropy. In Figure 8, the absolute conformational entropy for adsorbed native and reduced LTP is shown. As expected, in solution the reduced LTP has a conformational entropy that is higher than for the native LTP over the course of the simulation. We have already mentioned that conformational entropy acts against chain adsorption, and any factor that increases chain entropy will make adsorption more difficult. In the case of the reduced LTP, this manifests itself as a tendency for the protein chain to sit further away from the interfacial region. If we look at the density profiles for reduced protein and water in this system (Figure 7) we see that the maximum in the protein density profile for reduced LTP is indeed further from the interface than that for the native LTP (Figure 7). A second feature of the density profile for reduced LTP is that it appears to be sharper than that for the native LTP at the water-vacuum interface. This suggests that the reduced molecule may spread and flatten more at the interface than does the native molecule. Figure 3 shows the proportions of amino acid residues in helical and random coil

regions for the reduced LTP simulation run. The removal of four -S-S- bonds leads to a tendency for the reduced LTP to lose helical structure in solution. This is not surprising given the importance of the Cys48-Cys87 disulphide bond, in combination with the extended loop, to stabilizing the helical structure of LTP.26 In addition to this, there is evidence in Figure 3 that the reduced LTP forms new secondary structure at the interface in the form of small regions of β-sheet. This is not observed for the native LTP adsorbed to the water-vacuum interface. The significance of this will be discussed later. A plot of the RMSD for the reduced, adsorbed LTP molecule (Figure 4) further shows that the reduced molecule adopts an altered structure to the native LTP (the RMSD is higher for the reduced molecule). We can see from Figure 4 that the initial increase in RMSD is greater for the reduced LTP than for the native structure, which indicates a greater structural change for reduced LTP when it equilibrates with the water model. It is also apparent that after the initial rapid rise in RMSD, there is also a slower increasing trend up to 32 ns. Whether this is due



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Figure 7. Density profile for water molecules and protein across the simulation box in a vacuum-water interface system. The solid line represents water (–), and the dashed line represents protein (---). The water density across the box in the absence of the LTP molecule is shown as the dashed and dotted line (– · · – · · – · · ). (a) Native LTP; (b) reduced LTP. Figure 9. Snapshot conformation of (a) native LTP and (b) reduced LTP adsorbed at a decane-water interface after 40 ns (native LTP) and 20 ns (reduced LTP) of a MD simulation. Water molecules that occupy the space between the two decane interfaces have been removed for clarity. The global fold of the LTP is shown as a solid ribbon. Only adsorbed amino acid residue side chains are shown. Amino acid residues that are adsorbed in the interfacial region are highlighted in black.

Figure 8. Absolute conformational entropy over the course of the simulation for native and reduced LTP adsorbing at the water-vacuum and decane-water interfaces. – ) Native LTP in a water box; · · · · · · ) native LTP at the water-vacuum interface; – – – ) reduced LTP at the water-vacuum interface; ---- · ) native LTP at the decane-water interface; and – · · – · · – · · ) reduced LTP at the decane-water interface.

to a slow adsorption-induced unfolding or a slow relaxation of the reduced structure is not clear. To determine the cause of this slow change in RMSD requires longer simulations and further study. If we consider the radius of gyration for the adsorbed, reduced LTP (Figure 5), we can see that the reduced LTP has a slightly higher Rg than the native LTP. The components of Rg also show a change, with Rgz exhibiting a decrease, while both Rgx and Rgy increase, that is, the molecule appears to flatten slightly at the interface, which supports the observation that the density profile is sharper for the reduced LTP. Analysis of the amino acid residues in the adsorbed layer (Figures 6 and 2b) reveals that, in general, the same two domains are adsorbed in the reduced LTP as for the native LTP (Figure

6). The number of amino acid residues adsorbed is lower for the reduced LTP, however, and at the end of the 32 ns of the simulation no more than 18 residues are in the interfacial region. The proportions of hydrophilic, hydrophobic, and neutral side chains in the interfacial region again show that there is no preferential adsorption of hydrophobic amino acids, which suggests minimal conformational unfolding at the surface. The adsorbed residues for the reduced LTP are found in helix A, helix D, loop 1, and loop 3. Native LTP at the Decane-Water Interface. We can compare the LTP conformation at the water-vacuum interface with that at the water-decane interface. Figure 9a is a snapshot conformation after 40 ns of a simulation of the adsorption of native LTP at the decane-water interface. The first point to notice is that at the decane-water interface the LTP molecule penetrates into the decane phase (Figure 9a), while there is no penetration into the vacuum region at the vacuum-water interface (Figure 2a). Considerable penetration is seen even after 2 ns, and the LTP moves further into the decane layer as the simulation progresses. This observation is consistent with the Monte Carlo simulation studies of Anderson et al.43 who have studied adsorption of globular protein-like lattice chains at the oil–water interface. They observed an increase in entropy of the chains upon adsorption. This suggests that the chains are able to enter the oil phase, which allows them to adopt a greater number of conformations than they can access in the native state

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Figure 10. Density profile for water, protein, and decane across the simulation box in a decane-water interface system. The water density is shown as a solid line (–), protein density as a dotted line ( · · · · · · ), and decane density as a dashed line (----). (a) Native LTP; (b) reduced LTP.

in water. This leads to an increase in conformational entropy rather than the decrease expected when a protein is bound to a surface. From Figure 8 we can see that in our simulations adsorption of native LTP to the water-decane interface leads to a decrease in entropy compared to adsorption at the water-vacuum interface. This does not support the hypothesis that proteins are more flexible at the decane-water interface. We should note, however, that the entropy for LTP at the decane-water interface is still increasing, and we cannot rule out the possibility that it will eventually increase above that of the native LTP at the water-vacuum interface. We should remember that since the viscosity of the decane layer is higher than the water phase, the kinetics of relaxation of LTP in the decane phase will be slower than in the water phase of the water-vacuum interface. Experimental studies by Murray et al.44 also suggest that proteins are more unfolded and possibly more flexible at the oil–water interface. Further evidence for the penetration of LTP into the decane phase comes from the density profiles for oil, water, and protein in the system. In Figure 10, the penetration of protein into the oil phase is clearly visible where the density profiles for decane and protein overlap. This can be compared to the case where LTP is adsorbed to the water-vacuum interface, Figure 7, where LTP adsorbs to the interface, but stays only in the water phase and does not enter the vacuum space to any significant degree. It is also evident from Figure 10 that there is significant mixing of the decane and water phases in the interfacial region, because the density profiles for decane and water also overlap. The penetration of LTP into the oil phase is also accompanied by changes in the conformation of the LTP molecule. Figure 4 shows the RMSD for LTP molecules adsorbed to a decane-water interface. Unlike LTP adsorbed at the water-vacuum interface (Figure 4), at the water-decane interface there is a significant

Euston et al.

increase in RMSD after the LTP has adsorbed to and penetrated into the decane layer, indicating conformational rearrangement. This conformational change of LTP at the decane-water interface is accompanied by a change in the molecular dimensions of the protein (Figure 5). The radius of gyration (Rg) for adsorbed LTP in Figure 5 shows an increase over the course of the simulation at the decane-water interface, which is greater than the slowly increasing Rg observed at the water-vacuum interface. This suggests that conformational change may be greater at the decane-water interface. It should be noted that in both Figure 4 and Figure 5 the RMSD and Rg for the LTP at the decane interface does not appear to have reached equilibrium after 40 ns. We cannot rule out the possibility of further changes in the LTP conformation at longer timescales. At the decane-water interface the increase in Rg appears to be due mainly to an elongation in the z-direction (the component of Rg in the z-dimension, Rgz increases), possibly as the LTP molecule “squeezes” between the decane molecules as it enters the oil phase. Qualitatively, similar behavior is observed for lysozyme when it interacts with a lipid bilayer. Yuan et al.45 found that as the lysozyme penetrated the lipid bilayer the alkane chains of the constituent phospholipids become stretched due to steric crowding. Conversely, at the vacuum-water interface there is a contraction of the LTP molecule in the z-direction as the molecule resists movement into the vacuum region. LTP is a highly hydrophobic protein. It is known to bind lipids in vitro, although its in vivo function has not been identified with any certainty.46 It is, therefore, not surprising that it has a high affinity for an oil surface. In addition to the changes in conformation of the LTP molecule, we have also followed changes in the secondary structure through the adsorption process. Figure 3 shows the change in helix, sheet, and random coil residues as a function of simulation time. The secondary structural features of LTP are relatively stable at both the vacuum-water and decane-water interfaces. The proportion of helical and random coil amino acid residues remains fairly constant at around 50 and 40 residues, respectively. At the water-decane interface, native LTP does form some sheet structure, albeit intermittently, at times longer than 20 ns (Figure 3). This would appear to form from random coil residues as there is a concomitant decrease in random coil residues when sheet structure is formed (Figure 3). This was also observed for reduced LTP at the water-vacuum interface but not for the native LTP at the same interface (Figure 3). Adsorption of native LTP to the water-decane interface is accompanied by a displacement of water from the decane surface, as can be observed in the density profiles for protein, decane, and water (Figure 10). Adsorption and penetration of LTP into the decane layer also leads to changes in the interaction between amino acid residues and water molecules. As LTP enters the decane phase, there is a decrease in protein-water H-bonds and an increase in protein–protein H-bonds (data not shown). Presumably this is a result of dehydration of the part of the LTP molecule in the decane phase. In contrast, we found that LTP adsorbed at the vacuum-water interface showed no changes in protein–protein interactions throughout the 23 ns of the simulation. In addition, the numbers of protein–protein H-bonds are lower in LTP adsorbed at the vacuum-water interface than for LTP at the decane-water interface. Similarly, the numbers of protein-water H-bonds are higher for LTP at the vacuum-water interface than for LTP at the decane-water interface, presumably because less of the protein surface (and interior) is in contact with the water phase in the latter system. This dehydration of the protein surface may provide an extra


driving force for LTP adsorption and penetration into the decane layer in addition to water displacement from the interfacial region. Analysis of the amino acid residues that are adsorbed at the interface (Figures 6 and 9a) again reveals a preference for adsorption of two regions of the molecule: one is sited at the N-terminal end and involves adsorption of residues in or close to the block Leu14–His35, while the second extends from the C-terminal end and involves adsorption of residues in the block His59-Tyr91. Comparing the number of amino acid residues adsorbed into the interfacial regions at the water-vacuum and water-decane interfaces, we find, as expected, that there are more amino acid residues at the oil interface, where around half of the amino acids are associated with the interfacial layer or are completely in the decane phase, compared to approximately a quarter at the water-vacuum interface. The types of amino acid residues adsorbed at the water-decane interface are fairly evenly distributed between the hydrophobic, hydrophilic, and neutral side chain. From Figure 6, it is also evident that there are more residues from the LTP binding pocket (approximately half) residing in the interfacial region at the water-decane interface. The majority of residues adsorbed at the decane-water interface are found in helix A, helix D, loop 1, loop 3, and the extended loops region. Examination of the conformation of native LTP adsorbed at the decane-water interface reveals some interesting features. There appears to be a distinct alignment of the helical regions of the molecule and the extended loop parallel to the interface. Helix D has penetrated fully into the decane layer, with the C-terminal extended loop sitting parallel to this and just penetrating the decane layer. Helices B and C also lie in an extended conformation parallel to the interface, but both are in the water phase, with only some of the residues from helices B and C entering the decane layer. Helix A is the only helix that does not adopt an extended conformation parallel to the interface. Reduced LTP at the Decane-Water Interface. Adsorption of the reduced LTP at the decane-water interface also leads to penetration of the molecule into the decane layer. Figure 9b is a snapshot conformation after 20 ns of a simulation of reduced LTP adsorbing at the decane-water interface. We can see that the reduced LTP in Figure 9b adopts a more open conformation at the interface. This is confirmed if we look at the conformational entropy of the LTP chain given in Figure 8. The adsorbed, reduced LTP has a conformational entropy higher than for native LTP at the same interface but lower than for reduced LTP at the vacuum-water interface. Although the entropy for reduced LTP at the oil–water interface is lower than at the water-vacuum interface, the entropy at the oil surface is increasing faster than at the water-vacuum interface. Therefore, again, it is possible that the molecule is relaxing more slowly at the decane interface, and eventually, the entropy curves will cross over and the decane surface adsorbed LTP will indeed have the higher entropy when equilibrium is reached. In addition, analysis of the regions of the protein adsorbed to the decane interface (Figures 6 and 9b) reveals a broadly similar distribution to that seen for the native LTP in Figure 6. Parts of helix A, helix B, helix D, and loops 1 and 3 enter the decane phase, while in contrast to the native molecule, no residues from the extended loop are found in the decane. The preference of the extended loop for the water phase may be due to the breakage of the -S-S- bond between Cys48-Cys87, which stabilizes the hydrophobic contacts between the extended loop and the helices. When this is absent, the extended loop will become more flexible and may not enter the decane layer for entropic reasons.


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The conformational entropy for native LTP at the decane-water interface is lower than for the reduced LTP (Figure 8) and is probably a consequence of the greater penetration of the native LTP into the decane layer. This is also evident if we compare the density profiles for native LTP and reduced LTP (Figure 10) at the oil–water interface. We can see that the native LTP does indeed penetrate further into the decane layer. We can compare the conformational changes of reduced LTP at the decane- and vacuum-water interfaces by following the RMSD and Rg as a function of simulation time. The RMSD for reduced LTP at the vacuum-water interface and at the decane-water interface is shown in Figure 4. The evolution of RMSD is similar for LTP at both interface types. Compared to the native LTP at the decane-water interface (Figure 4), we see less conformational change for the reduced LTP. The similarity of RMSD for reduced LTP at both interfaces suggests that most of the conformational change occurs due to the increase in flexibility brought about by breaking the disulphide bonds. The radius of gyration for reduced LTP at both decane- and vacuum-water interfaces are shown in Figure 5. At both interfaces, there is a flattening of the LTP molecule, as it adsorbs to the surface. This is more pronounced for the decane-water interface where the component of Rg normal to the interface (Rgz) is substantially smaller than the components in the plane parallel to the interface (Rgx and Rgy). The behavior of reduced LTP at the decane surface contrasts to that of native LTP, where an elongation of the molecule in the z-direction was observed. It would seem, therefore, that changes in the flexibility of the LTP structure have an influence on the conformation adopted at the decane-water interface. In Figure 3, the proportions of helix, sheet, and random coil residues for the reduced LTP at the decane-water interface are shown. At the decane-water interface the proportions of helix and random coil stay approximately the same, although there may be a slight trend for increasing helix as the simulation progresses. No sheet structures are formed over the course of the simulation at the decane surface for reduced LTP. At the vacuum-water interface, the proportions of helix and random coil show only small fluctuations for the first 10 ns of the simulation and then start to fluctuate more widely after this time (Figure 3). This also corresponds to the time at which we begin to see formation of sheet structures in the adsorbed, reduced LTP molecule. Further analysis of the sheet structures for the reduced LTP at the vacuum-water interface and the native LTP at the decane-water interface, using the Swiss PdbViewer,38 show that it is the same six residues that are involved in sheet formation in both systems. These correspond to the amino acid triplets Val(16)-Gln(18)-Gly(19) and Asn(60)-Leu(61)Asn(62). Of these two triplets, the second (Asn,Leu,Asn) is part of the random coil region in loop 3 in the native molecule, while in the first triplet, the Val and Gly residues are at the terminal end of helix A. Analysis of conformations for native LTP at the decane-water interface and reduced LTP at the vacuum-water interface that contain these β-sheet structures has confirmed that the six residues involved in formation of the sheet are found in the interfacial region. Analysis of the positions of the six residues reveals that the structure formed is the antiparallel β-sheet. Like the adsorption of native LTP, adsorption of reduced LTP is also accompanied by changes in the hydrogen bonding in the system. On average, the numbers of protein–protein H-bonds are similar at both interfaces, suggesting that the conformational changes caused by cleavage of -S-S- bonds dominate over any conformational change induced by adsorption at the decane-water

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interface. The numbers of protein-water H-bonds are slightly lower for LTP at the decane surface, reflecting the dehydration of the protein structure caused by penetration of the LTP into the oil phase. Several experimental studies have produced results that support the simulation studies. Lu et al.47,48 have shown that lysozyme (a protein with a rigid structure) adopts a more spread out conformation at a hydrophobic surface47 than at a hydrophilic surface.48 They suggest that this is due to water-surface interactions being more favorable at hydrophilic surfaces and, therefore, displacement of water less likely. Other authors have also noted changes in secondary structure when proteins adsorb to a surface. Lefevre and Subirade10 have noted that the globular milk protein β-lactoglobulin forms intramolecular β-sheet when adsorbed at an oil–water interface. This occurs when the β-barrel structure of β-lactoglobulin unfolds upon surface denaturation and then reforms intramolecularly. An increase in intramolecular R-helical content has also been observed for β-lactoglobulin adsorbed to hydrophobic surfaces.49–51 Raffaini and Ganazzoli52,53 have simulated the adsorption of peptide domains of albumins52 and fibronectin.53 The fibronectin fragments contained predominantly R-helix, while the albumin domains only β-sheet. The β-sheet regions were observed to be more stable to adsorptioninduced unfolding than the helical regions. This is supported by the few MD simulations that have been carried out for small whole globular proteins at solid surfaces54 and lipid bilayers.55 Norde and Lyklema56 have hypothesized that unfolding of protein secondary structure gives an increase in chain entropy that is sufficient to compensate for both the loss of enthalpy due to breakage of bonds holding together the secondary structure and the loss of chain conformational entropy. Another study suggests that the changes in secondary structure upon adsorption may depend on the protein. Lad et al.57 found that when lysozyme adsorbs to the air–water interface, it forms an unfolded layer with a high antiparallel β-sheet content and that this occurs within 10 min of adsorption. Bovine serum albumin and β-lactoglobulin on the other hand, although they too are observed to adsorb rapidly, show little change to their native secondary structure.57 The unfolding of secondary structure at the interface is unlikely to be a simple process. The relative (and absolute) stabilities of helix and sheet regions of protein molecules will most likely depend on factors such as the solution pH and ionic strength, the nature and structure of surrounding peptides, the presence of disulphide bonds, and the nature of the surface.

Conclusions The simulations presented here have been carried out over ns time scales that are very short compared to the time scale of protein conformational changes at interface which may take minutes or hours. However, we are encouraged that some conformational changes in adsorbed LTP can be observed at these short times. Native LTP adsorbing at both water-vacuum and decane-water interfaces undergoes some change in tertiary conformation, which is greater at the decane surface. This appears to be due to the ability of the LTP molecule to penetrate some way into the decane layer, while at the water-vacuum interface the molecule remains wholly in the water phase. Our simulations also demonstrate the effect of changing molecular flexibility on the adsorption of proteins. When the LTP molecule is fully reduced by removing the four disulphide bonds, the increase in flexibility of the molecule leads to an increased conformational entropy, which reduces the affinity of the

Euston et al.

molecule for the interfacial region. Although the LTP molecule does appear to spread at the interface, we see no evidence of unfolding of the secondary structure (helical regions for LTP) or surface denaturation to expose the hydrophobic core of the molecule. However, what we do see is formation of a new β-sheet secondary structure for native LTP adsorbing at the decane interface and reduced LTP at the water-vacuum interface. The formation of β-sheet structure is a phenomenon that has been observed experimentally in other proteins.10,57 Our simulations indicate that similar regions of the LTP molecule surface adsorb to both the decane-water and water-vacuum interfaces. In particular parts of helix A, loop 1, helix B, loop 3, and helix D appear to be important in the adsorption process. Currently, our longest simulation is 40 ns. To move into the µs and perhaps the ms timescales will require the use of accelerated MD techniques. Several such methods have been proposed and are either being developed or have been demonstrated for use with peptides and proteins.58–65 These include hyperdynamics,58,59 temperature accelerated dynamics (TAD),60,61 metadynamics,62,63 parallel replica MD,64 and essential dynamics65 with flooding potentials.66 Work using accelerated dynamics methods is already under way in our laboratories. Acknowledgment. The authors acknowledge the EPSRC for access to the CSAR HPC facility, the National Service for Computational Chemistry Software (http://www.nsccs.ac. uk) for access to COLUMBUS HPC facilities and HeriotWatt University Computer Centre use of a DEC Alpha server and the HWU central HPC facility.

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Comparison of the Adsorbed Conformation of Barley Lipid Transfer

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