ARTICLE pubs.acs.org/JPCB
Secretory Phospholipase A2 Activity toward Diverse Substrates Jesper J. Madsen,† Lars Linderoth,†,^ Arun K. Subramanian,†,§ Thomas L. Andresen,‡ and G€unther H. Peters*,†,§ †
Department of Chemistry and ‡Department of Micro- and Nanotechnology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark § MEMPHYS-Center for Biomembrane Physics
bS Supporting Information ABSTRACT: We have studied secretory phospholipase A2-IIA (sPLA2) activity toward different phospholipid analogues by performing biophysical characterizations and molecular dynamics simulations. The phospholipids were natural substrates, triple alkyl phospholipids, a prodrug anticancer etherlipid, and an inverted ester. The latter were included to study head groupenzyme interactions. Our simulation results show that the lipids are optimally placed into the binding cleft and that water molecules can freely reach the active site through a well-defined pathway; both are indicative that these substrates are efficiently hydrolyzed, which is in good agreement with our experimental data. The phospholipid analogue with three alkyl side chains forms aggregates of different shapes with no well-defined sizes due to its cone-shape structure. Phosphatidylglycerol and phosphatidylcholine head groups interact with specific charged residues, but relatively large fluctuations are observed, suggesting that these interactions are not necessarily important for stabilizing substrate binding to the enzyme.
1. INTRODUCTION Phospholipases A2 (PLA2s: EC 3.1.1.4) are enzymes that specifically hydrolyze the sn-2 fatty acid acyl ester bond of aggregated glycerolphospholipids, producing free fatty acid and lysophospholipid.15 PLA2s are found both intra- (cytosolic) and extracellularly (secreted) in mammalian tissues, and the PLA2 superfamily includes the calcium-independent PLA2, the high molecular weight cytosolic PLA2, and the low molecular weight secretory PLA2 (sPLA2).1,6,7 Secretory PLA2s are homologous, and at least 10 distinct members (IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XIIA) have been identified.4,5,8,9 All of these phospholipases share the same catalytic mechanism in which a calcium ion plays an essential catalytic role.10,11 The calcium ion coordinates to the substrate, an aspartate, and a main-chain carbonyl oxygen. The active site consists of a dyadic His/Asp pair and resembles the triad (Ser/ His/Asp) found in proteases.12,13 In sPLA2s, a water molecule acting as the nucleophile takes the same role as Ser in proteases.12 The His/Asp pair abstracts a proton from an incoming water molecule, which then acts as the nucleophile in the attack on the sn-2 position of the carbonyl carbon in the phospholipid. This leads to the formation of a tetrahedral intermediate,1417 and subsequently, the sn-2 oxygen is protonated in concert with the productive collapse of the tetrahedral intermediate, releasing the products fatty acid and lysophospholipid.8,10,18,19 A common characteristic for phospholipases is that their activity increases substantially when the lipid concentration is above the critical micelle concentration,10,2023 a phenomenon known as interfacial activation.24 However, different subtypes of enzymes show different specificity toward lipid substrates.25,26 For instance, the sPLA2-IIA subtype r 2011 American Chemical Society
is mainly active toward anionic phospholipids,2527 whereas the sPLA2-V and -X subgroups catalyze the hydrolysis of both anionic and zwitterionic phospholipids.26,28 It has been suggested that this is controlled by the difference in the amino acids located on the interfacial binding surface (i-face; interfacial specificity) rather than the substrate affinity controlled by the active-site residues (catalytic site specificity).9,22,24,2933 Among the different secretory phospholipases A2, sPLA2 group IIA and group V have drawn much attention due to their important role in inflammatory diseases.34,35 sPLA2-IIA is expressed in a variety of human tissues and cells,36,37 while sPLA2-V is found in human heart, placenta, lung, liver, and neutrophils.38,39 Secretory sPLA2-IIA has been implicated in several inflammatory conditions,4044 where it hydrolyses phospholipids, leading to arachidonic acid that is converted by downstream metabolic enzymes to various bioactive lipophilic compounds known as eicosanoids, including prostaglandins and leukotrienes.34,35,45 The hydrolysis products, fatty acid and lysophosphatidylcholine, are also important precursors of other lipid mediators such as platelet-activating factor.4 Elevated expression of sPLA2-IIA has also been reported in several types of malignancies, including, for instance, pancreatic, breast, and prostate cancer.4654 This observation has been explored in a novel drug delivery system,55 where the high levels of sPLA2 are used as a site-specific triggering mechanism for anticancer drug delivery by liposomal carriers.5557 Secretory Received: December 21, 2010 Revised: April 19, 2011 Published: May 11, 2011 6853
dx.doi.org/10.1021/jp112137b | J. Phys. Chem. B 2011, 115, 6853–6861
The Journal of Physical Chemistry B PLA2-IIA hydrolyses the phospholipids composing the liposomes and thereby destroying the integrity of the liposomes. This results in the release of the cargo (anticancer drug). Additionally, the hydrolysis products, free fatty acid and lysophosphatidylcholine, also display a synergistic effect as permeability enhancers, resulting in an increase of drug concentration in the cancer cell.56,58 One of the challenges in developing an efficient drug delivery system is to optimize the liposomal drug carrier characteristics,55 which, for instance, include drug permeability, release profiles, and pharmacokinetic properties of the carriers. Liposome properties can be tuned by modifying lipid composition or by using new (unnatural) sPLA2-degradable phospholipid analogues in the liposome formulation.56,57 Here, we are interested in designing new phospholipid analogues that form stable liposomes and are effectively hydrolyzed by sPLA2IIA. We have previously shown that sPLA2-IIA can efficiently hydrolyze unnatural phospholipid analogues with the head group in the 2-position59 and tolerate certain substrates with substitution of a relatively small side chain located at the sn-1 position.60 Although transition-state studies have established the basis for understanding enzyme kinetics,6169 we could show that the steps prior to the transition state can also critically determine the outcome of the enzymatic reaction.59 The phospholipid analogues are essentially limiting the space available in the binding pocket of sPLA2 and thereby reducing the access of water molecules to the catalytic site.59,7074 In the current study, we have tested different phospholipid analogues to increase our understanding on how structural modifications in phospholipid analogues affect the activity profile of sPLA2. The first two analogues have an extended side chain in the sn-1 position (R- and S- enantiomers). The design of these analogues was motivated by our previous study, which showed that relatively short extension of the sn-1 side chain interfered with an incoming water molecule that acts as the nucleophile in the enzymatic reaction. If the sn-1 side chain is sufficiently long, it should not be able to enter the binding pocket, and consequently, water molecules should have free access to the active site cavity. The other phospholipid analogue is an inverted ester that has been explored in drug delivery systems.75 We additionally studied whether the head group (phosphatidylcholine versus phosphatidylglycerol) will utilize specific interactions with amino acids in the enzyme and thereby affect accessibility of water molecules to the catalytic site.
2. EXPERIMENTAL METHODS 2.1. Preparation of Vesicles. Phospholipids were hydrated in an aqueous buffer (150 mM KCl, 10 mM HEPES, 30 mM CaCl2, 10 mM EDTA, pH = 7.5). Multilamellar vesicles (MLVs) formed spontaneously. To ensure complete hydration, the lipids were hydrated for 1 h at 65 C. During the hydration, the lipids were vortexed every 15 min. When small unilamellar vesicles (SUVs) were needed, the MLVs were sonicated for 1 h at 5 C above the lipid main-phase transition temperature. The total lipid concentration in the buffer solutions was 2 mM. 2.2. Differential Scanning Calorimetry (DSC). DSC of 2 mM MLVs was performed by using a Microcal MC-2 (Northhampton, MA, U.S.A.) ultrasensitive power compensating scanning calorimeter equipped with a nanovoltmeter. The scans were performed in the upscan mode at a scan rate of 10 C/h. An appropriate baseline was subtracted from the calorimeter curves to afford the melting enthalpies of the MLVs.
ARTICLE
2.3. Activity Measurements. The conditions used to perform the sPLA2 activity measurements were as follows: 0.15 mM phospholipid as SUVs, 150 nM sPLA2, 0.15 M KCl, 30 mM CaCl2, 10 mM EDTA, and 10 mM HEPES (pH 7.5). The catalytic reaction was initiated by addition of 8.9 mL of a 42 mM snake (Agkistrodon piscivorus piscivorus) venom sPLA2 stock solution to a 2.5 mL SUV suspension thermostatted at the main-phase transition temperature of the vesicles prior to the addition of the enzyme. The time-dependent activity of sPLA2 was monitored from the changes in the 90 static light scattering, giving information of changes in the lipid morphology as nonbilayer-forming lysophopholipids and fatty acids were generated. High-performance liquid chromatography (HPLC) and MALDI-TOF MS were used to monitor the enzymatic reaction by collecting samples at different time intervals, as described previously.70,76 Fluorescence measurements were performed using an SLM DMX 1100 spectrofluorometer. Purified snake venom PLA2 was a generous gift from Dr. R.L. Biltonen (University of Virginia, VA, U.S.A.). 2.4. Sequence Conservation Analysis. The conservation of amino acid residues in the sPLA2 family was analyzed using the ConSurf77,78 web server. Conservation scores are based on an evolutionary relationship between the target protein and its homologues through an empirical Bayesian method,79 and the scores range from 1 (poor conservation) to 9 (high conservation). The multiple-sequence alignment was performed with a set of 309 homologues, which were fetched through a single-iteration PSIBLAST80 search with an E-value cutoff of 0.001. 2.5. Molecular Dynamics Simulations. The crystal structures of bee-venom (Apis mellifera) phospholipase A2 complexed with the transition-state analogue, L-1-O-octyl-2-heptylphosphonylsn-glycero-3-phosphoethanolamine (diC8(2Ph)PE),3,10 resolved to 2.0 Å and human sPLA2-IIA complexed with 6-phenyl-4(R)(7-phenyl-heptanoylamino)-hexanoic acid81 resolved to 2.1 Å were obtained from the Protein Data Bank82 (entry codes: 1poc and 1kqu, respectively). The initial modeling step involved placing diC8(2Ph)PE in the binding cleft of sPLA2-IIA, as described previously.59,70 The structures of the different phospholipid analogues were built from diC8(2Ph)PE using SPARTAN version 1.0.2 (Wavefunction Inc., Irvine, California, U.S.A.). The structures are shown in Figure 1A, and abbreviations for the phospholipid analogues are defined in Table 1 and used throughout the text. Missing parameters for (R)-inverted ester and (S)- and (R)-triple alkyl were taken from the Charmm27 parameter set describing similar atom types (Figure S1, Supporting Information). Parameters for the ether group in the prodrug anticancer etherlipid (ProAEL) were taken from the Charmm32 ether force field. The different enzymephospholipid complexes were solvated using the program SOLVATE (by Grubm€uller).83 Eighteen water molecules were randomly replaced with chloride ions to neutralize the systems. The final system contained ∼5000 water molecules. For all simulations, NAMD84 was used with the Charmm27 all-atom parameter set and the TIP3P water model.85 Simulations were performed for 10 ns, and in order to assess the statistical uncertainties, simulation for each complex was repeated three to seven times (Table 1). Different initial conditions were obtained by varying the number of steepest-descent energy minimization steps in intervals of 250 steps, that is 250, 500, 750, and so forth minimization steps. This was followed by an initial simulation of 100 ps for heating the systems to T = 300 K. Simulations were carried out in the NPT ensemble, that is, at a constant number of atoms (N), pressure (P), and temperature (T). An isotropic constant ambient pressure of 1 atm was imposed 6854
dx.doi.org/10.1021/jp112137b |J. Phys. Chem. B 2011, 115, 6853–6861
The Journal of Physical Chemistry B
ARTICLE
Figure 1. (A) Chemical structure of the phospholipid analogues. (B) Schematic representation of the catalytic mechanism of sPLA2 and part of the hydrogen-bonding network are shown. Atom types given in parentheses refer to Protein Data Bank nomenclature. The overlap of the two circles (transparent region) indicates the HS region.
Table 1. Summary of the Simulations Carried outa number of short nameb
length of simulations root mean square displacement mean (
simulations
(ns)
SDc (Å)
(R)-1,2-dipalmitoyl-glycero-3-phosphocholine PC (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phoshocholine ProAEL
6 3
each 10 each 10
1.4 ( 0.2 1.4 ( 0.1
(R)-1,2-dipalmitoyl-glycero-3-phosphoglycerol
PG
5
each 10
1.4 ( 0.2
(20 R,2S)-2,3-dihydroxypropyl
(R)-inverted ester
7
each 10
1.2 ( 0.1
(S)-triple alkyl
6
each 10
1.5 ( 0.3
(R)-triple alkyl
10
each 10
1.4 ( 0.2
name
20 -octadecanoyloxy-50 -hexadecyloxy-50 -oxopentan-10 -yl phosphate (2S,20 S,3S)-2,3-dihydroxypropyl 20 ,30 -octadecanoyloxy-nonadecyl phosphate (2S,20 S,3R)-2,3-dihydroxypropyl 20 ,30 -octadecanoyloxy-nonadecyl phosphate a
The last column lists the average root-mean-square displacement (rmsd) of CR atoms with respect to the protein structure after minimization. Means and their standard deviations (SDs) are based on a series of simulations of a particular complex. b Abbreviation used in the text. c SD = standard deviation 2 1/2 calculated as SD = [(∑N ; N = number of simulations of a particular sPLA2phospholipid complex. i=1 (xi x) )/(N 1)]
using the Langevin piston method86 with a damping coefficient of 5 ps1, a piston period of 200 fs, and a decay of 500 fs. A time
step of 1 fs was used throughout. The particle mesh Ewald summation method was used for computation of the electrostatic 6855
dx.doi.org/10.1021/jp112137b |J. Phys. Chem. B 2011, 115, 6853–6861
The Journal of Physical Chemistry B forces87,88 with a grid spacing of ∼1.0 Å, and a fourth-order spline was used for the interpolation. The long-ranged part of the electrostatic forces was evaluated every fourth femtosecond. van der Waals interactions were cut off at 12 Å in combination with a switching function starting at 10 Å. Periodic boundary conditions were applied in x, y, and z directions. The analyses of the trajectories were performed using the Visualization Molecular Dynamics software suite.89 2.6. Water Count and Trajectory Analysis. A prerequisite for successful hydrolysis is that a water molecule acting as a nucleophile can enter the catalytic cleft. The catalytic mechanism has been identified by X-ray crystallography revealing, that the catalytic device of sPLA2 is essentially characterized by an aspartic acid histidine (Asp91His47) dyad, a calcium-binding site, and a water molecule acting as the nucleophile.2,3 A schematic representation of the catalytic mechanism is shown in Figure 1B and indicates that the calcium ion (cofactor) is coordinated to the Asp48 carboxylate groups (atoms: OD1 and OD2), the Gly29 carbonyl oxygen (O), and the carbonyl (O22) as well as the phosphate oxygen (O) in the substrate (S). Atom types given in parentheses refer to the Protein Data Bank nomenclature. Asp91 forms hydrogen bonds with Tyr51 and Tyr66. The water molecule acting as the nucleophile enters the region between His47(ND1) and S(C21). This region will be hereafter referred to as the HS region (Figure 1B). In order to quantify the probability that water molecules enter the HS region, we counted the number of water molecules within certain distances (d = 36 Å; Δd = 0.5 Å) from both His47(ND1) and S(C21). Because the motion of the water molecules appears random (stochastic) in nature, there was a significant spread in the water counts. Therefore, we normalized the counts for each sPLA2lipid complex by dividing the respective water counts by the water count determined at 6 Å. As previously shown, this analysis provides a suitable tool to predict whether a new phospholipid analogue will successfully be hydrolyzed by sPLA2.59,70,74 For convenience, we refer to a water molecule that reached the HS region and was e3.5 Å away from both His47(ND1) and S(C21) as H2OHS. Using a similar approach as that proposed by Garcia and Hummer,90 we identified and back-tracked H2OHS molecules in each simulation. To map the entry path, we calculated the distance between the oxygen atom of H2OHS and any atom of a particular residue. From these data, we calculated the frequency of H2OHSamino acid contacts using a cutoff distance of 3.5 Å.
3. RESULTS AND DISCUSSION We have performed a series of MD simulations of human sPLA2IIAphospholipid complexes (Table 1) to (i) study the binding mode of these substrates, (ii) identify key interactions between the head groups (phosphoglycerol versus phosphocholine) with surrounding residues, and (iii) identify if (R)-inverted ester, (S)-triple alkyl, and (R)-triple alkyl are potential substrates for sPLA2. In the last section, we compare the simulation results with our biophysical characterization. 3.1. sPLA2Phospholipid Complex. MD simulations of the different sPLA2phospholipid complexes were performed three to seven times depending on the complex (Table 1). The stability of the MD simulations of the sPLA2substrate complexes were checked by calculating the root-mean-square deviations (rmsds) of the CR atoms of the amino acids compared to the initial protein structure. The complexes reached a constant rmsd value within 2 ns (Figure S2, Supporting Information), and the rmsd averaged
ARTICLE
Figure 2. (A) 2D scatter plots of distances between selected atoms that were chosen according to their importance in the calcium-dependent enzymatic reaction.10 The distances were extracted from representative simulations of sPLA2PG (black), sPLA2(R)-inverted ester (red), sPLA2(R)-triple alkyl (green), and sPLA2(S)-triple alkyl (blue) complexes. (B) Mean distances of His47(HE2)Asp91 (OD1), His47(ND1)S(C21), Ca2þGly29(O), S(O22)Gly29(NH), and S(O22) Ca2þ extracted from the simulations of different sPLA2substrate complexes as defined in the inset. Distances are averages over time and simulations of each substrate. See Figure 1B for identification of the distances in the binding pocket.
over time and trajectories for each complex were in the range of 1.21.5 Å (max. standard deviation (0.3 Å) (Table 1). For successful hydrolysis, the MichaelisMenten complex has to be stable, and water molecules acting as the nucleophile in the enzymatic reaction have to enter the active site cavity. We first considered the MichaelisMenten complex and the key distances involving the cofactor calcium, the substrate, or residues as part of the catalytic machinery are illustrated in Figure 1B (dashed lines). These distances were monitored throughout the simulations. All phospholipids form stable complexes, and key distances in the MichaelisMenten complex are maintained throughout the simulations, as indicated by the 2D scatter plots (Figure 2A). Timeaveraged distances of His47(HE2)Asp91(OD1), His47(ND1) S(C21), Ca2þGly29(O), S(O22)Gly29(HN), and S(O22) Ca2þ extracted from the simulations are presented in Figure 2B. Atom types indicated in parentheses refer to the Protein Data Bank nomenclature. Numerical values are summarized in 6856
dx.doi.org/10.1021/jp112137b |J. Phys. Chem. B 2011, 115, 6853–6861
The Journal of Physical Chemistry B
ARTICLE
Figure 3. Relative water counts (RWCs) observed in the HS region using different radii from both His47(ND1) and S(C21). RWCs were determined by first dividing the individual RWCs by the RWC determined at 6 Å and then averaging RWC at a certain radius for each complex defined in the inset.
Table S1 (Supporting Information). Differences observed between the different complexes are within the statistical uncertainties. The first criterion (i.e., stable MichaelisMenten complex) is fulfilled, and we therefore focus on the second criterion — water accessibility to the active site region. We calculated the probability of water molecules reaching the region between His47(ND1) and S(C21), referred to as the HS region (Figure 1B; overlapping region of the two circles). Average relative water counts (RWCs) extracted from the simulations are displayed in Figure 3, and numerical values are provided in Table S2 (Supporting Information). Besides for (R)-inverted ester, the probability of observing a water molecule at certain distance from both His47(ND1) and S(C21) (corresponding to different sizes of the HS region) is within the statistical uncertainties. The RWC for (R)-inverted ester is approximately 10 times lower than the those calculated for the other lipids. In view of the relatively large statistical uncertainties in RWCs for the other phospholipids, we cannot rank the phospholipids according to sPLA2 efficiency toward these lipids. However, the results suggest that all phospholipid analogues are substrates for sPLA2. 3.2. Water Molecule Intake Pathway into the Active Site. We have previously shown that on the basis of a sequence conservation analysis, the residues Leu2 [7], Phe5 [9], Arg42 [8], Cys43 [9], Cys44 [9], His47 [9], Asp48 [9], Cys50 [9], Tyr51 [9], Cys90 [9], Asp91 [9], and Ala94 [8], which are located within 5 Å of the active site, are highly conserved. The conservation scores are given in square parentheses, where a conservation score of 9 reflects that the residue is highly conserved (nonconserved residues would have a score of 1). Leu2 plays an important role in substrate specificity in terms of a substrate’s acyl chain length.91 The cysteine residues are involved in SS bridges. His47, Asp48, and Asp91 are part of the catalytic machinery.2,3 On the basis of previously conducted simulations of sPLA2natural substrates and sPLA2chlorambucil prodrug complexes, we could show that only a few residues encountered frequent contact with incoming water molecules (unpublished data). We have extended the analysis to the current phospholipid analogues, where we have back-tracked the water molecules that have reached the HS region and determined the residues that had been in contact with the incoming water molecules. Averaged over H2OHS and simulations of the individual complexes, the normalized frequencies extracted from the sPLA2PG/(R)-inverted ester/
Figure 4. (A) Waterresidue interaction frequencies are shown for sPLA2PG, sPLA2(R)-inverted ester, sPLA2(S)-triple alkyl, and sPLA2(R)-triple alkyl complexes. The analysis was done as follows: Waters that reached the catalytic site were identified and back-traced to determine the residues with which they interact. A cutoff of 3.5 Å was used in the calculations. (B) Snapshots displaying the water intake mechanism. Protein is shown as a ribbon and colored green. H2OHS is displayed as a stick structure and colored yellow. The substrate is partly shown and displayed in stick structure and colored according to the atom type (C = green, O = red, P = gold, H = white). Residues that are within 3.5 Å of H2OHS are displayed in a stick structure and colored orange. Otherwise, selected residues are colored according to the atom type (C = green, N = blue, O = red, H = white). H2OHS enters the sPLA2 structure at Asn1, Leu2, and Lys62 (i). The next part of the water path is defined by Phe5, Tyr51, and Asp91 (ii). The water molecule further diffuses in the direction of His47/Tyr51 and finally in the HS region. Results are representative for all simulations (iii). 6857
dx.doi.org/10.1021/jp112137b |J. Phys. Chem. B 2011, 115, 6853–6861
The Journal of Physical Chemistry B
Figure 5. Histogram of selected distances between charged residues and head group atoms as well as the C-terminal residue Cys124. S refers to the substrate, and atom types correspond to the Protein Data Bank nomenclature. Distances were extracted from the combined trajectory of the (A) sPLA2PC simulations [a: Arg53(CZ)Cys124(C); b: Glu55(CD)S(N); c: Lys62(NZ)S(O3)] and (B) sPLA2PG simulations [a: Arg53(CZ)Cys124(C); b: Glu55(CD)S(HO2); c: Glu55(CD) S(HO3); d: Lys62(NZ)S(O3); e: Lys52(NZ)S(OG2); f: S(HO3) S(O1)].
(R)-triple alkyl/(S)-triple alkyl simulations are 0.06(0.02/ 0.05(0.02/0.08(0.07/0.04(0.03 (Asn1), 0.09(0.02/0.07(0.02/ 0.04(0.03/0.06(0.01 (Leu2), 0.08(0.04/0.04(0.03/0.11(0.09/0.05(0.05 (Phe5), 0.10(0.03/0.08(0.04/0.07(0.08/0.15(0.03 (His47), 0.17(0.02/ 0.13(0.02/0.16(0.12/0.23(0.07 (Tyr51), 0.03(0.01/0.03(0.01/ 0.02(0.02/0.04(0.01 (Thr61), and 0.07(0.04/0.06(0.03/0.02(0.02/ 0.07(0.03 (Lys62). These values are significantly larger than the ones observed for other residues (e∼0.01), and the same picture emerges. The surface-accessible residues Asn1, Leu2, Thr61, and Lys62 and residue Phe5, His47, Tyr51, Tyr66, and Asp91 located in the active site cleft have frequent contact with the incoming water molecules (Figure 4A). Asn1, Leu2, and Lys62 are surfaceaccessible, and the backbone of these residues forms a rim above a hydrophobic groove formed by Phe5, Ile9, Phe23, and Phe9810,15,22,92 (Figure 4B, snapshot i). Figure 4B displays the water molecule passage toward the HS region. Residues are displayed in stick structures and colored orange when any of their side chain atoms are within 3.5 Å of H2OHS. H2OHS is displayed in stick structures and colored yellow. Once H2OHS enters the groove (Figure 4B, snapshot ii), it passes Asp91 and encounters Phe5 and Tyr51 that form aromatic walls. Visual inspection of the water trajectories suggests that the aromatic rings divert the direction of incoming water molecules toward the HS region (Figure 4B, snapshot iii) and that the orientation of Phe5 is partly restrained by the highly conserved residue Ile9 and the phospholipid acyl chain, thus maintaining a defined size for the hydrophobic groove. The important role of these residues for enzyme activity and substrate selectivity has been established by mutational studies,91,9396 which showed that the aromaticity of Phe5 and Tyr51 is important for maintaining enzyme activity. For instance, the Tyr51 f Phe mutation resulted in reduced protein stability, but the activity of the mutant was similar to the wild-type enzyme.15,93 We may speculate that nonaromatic substitutions of these residues will disturb the water flow and hence reduce sPLA2 activity. The current results can also explain previously reported sPLA2 kinetic data for digestion of phospholipid analogues with relatively short sn-1 substitutions.70 In view of the present study, it becomes clear that substitutions at the sn-1 position can block the entrance of H2OHS into the hydrophobic groove.
ARTICLE
3.3. Effect of the Head Group. Crystallographic studies indicated that the moderately conserved residue Lys62 interacts with the sn-3 phosphate oxygen of transition analogues.97 This observation suggests that this interaction may contribute to substrate stabilization in the binding pocket. To gain further insight into the role of proteinhead group interactions, we monitored the interaction pattern of charged residues surrounding the lipid head group in the sPLA2natural substrate (PC; PG) simulations. The hydroxyl groups (HO2, HO3; Figure 1) in PG or the choline group (N; Figure 1) in PC are potential interaction sites of the head group to the closely located charged residues Arg53, Glu55, Lys52, and Lys62. Average distances between these charged groups and selected head group atoms are shown in Figure 5A (PC) and Figure 5B (PG), and their time evolutions are shown in Figures S3 and S4 (Supporting Information). Most significant interactions are observed between Glu55 and the hydroxyl groups (HO2, HO3) of the PG head group or the choline group (N) of the PC head group. Noticeable for both systems, Arg53 interacts strongly with the C-terminal residue Cys124. Additional in the sPLA2PG simulations, an intramolecular hydrogen bond between the hydroxyl group (HO3) and the phosphate group (O1) of the head group is observed (Figure 5B; Figure S4, Supporting Information). In both the sPLA2PC and PG simulations, Lys62SO3 interactions only play a minor role (Figure 5), and significant fluctuations are observed (Figures S3 and S4, Supporting Information), indicating that even though this interaction is observed in the crystal structure, it is not important for hydrolysis. The MD simulations are in agreement with experimental studies, where we have shown that human sPLA2IIA is able to hydrolyze both zwitterionic PC and negatively charged PG in DMPG/DMPC liposomes containing at least ∼36% PG.25,26,98 Hence, the negatively charged lipid is essential for attracting human sPLA2-IIA toward the liposome interface. Once absorbed, human sPLA2-IIA does not distinguish between PC and PG,26 demonstrating that the head groups choline versus glycerol do not affect the actual catalysis. The simulations indicate that all substrates are subject to sPLA2 hydrolysis. Biophysical characterization of liposomes consisting of PC, PG, and ProAEL has been reported.26,28,59,99 The studies indicated that these substrates form well-defined liposomes, and ProAEL is efficiently hydrolyzed by sPLA2. The other phospholipid analogues have not been characterized. In the following section, we present the biophysical characterization of (R)-inverted ester and (R)-triple alkyl. The organic synthesis of these compounds has been reported elsewhere.71 3.4. Biophysical Characterizations. The ability of the phospholipid analogues to form well-characterized liposomes was assessed by monitoring differential scanning calorimetry (DSC) scans in the temperature range of 1065 C. A sharp peak during a melting transition would indicate a cooperative melting of the lipids, that is, lipids are well-ordered in the liposomes. For (R)-triple alkyl, the DSC scan showed no melting transition (Figure S5A, Supporting Information), which indicates that the phospholipid analogue is not organized in a highly ordered fashion in the lipid bilayer. This is most likely caused by the shape of (R)-triple alkyl. The extra acyl chain disrupts the cylindrical shape of the phospholipids and probably induces a more cone-like structure that leads to the formation of inverted hexagonal phases.100 To further elucidate this, the solution was analyzed by dynamic light scattering (DLS), which showed that the sample contained structures with no well-defined sizes and with average diameters in the 1000 nm range (Figure S5B, 6858
dx.doi.org/10.1021/jp112137b |J. Phys. Chem. B 2011, 115, 6853–6861
The Journal of Physical Chemistry B
ARTICLE
Figure 6. (A) HPLC chromatogram illustrating the effect of sPLA2-catalyzed hydrolysis of (R)-triple alkyl. The chromatograms show the amount of phospholipid before the addition of sPLA2 (0 h) and 24 h after the addition of sPLA2. (B) Comparison between the sPLA2-mediated hydrolysis of (R)inverted ester monitored by MALDI-TOF MS and HPLC. MALDI-TOF MS spectra for (R)-inverted ester are provided in Supporting Information (Figure S6).
Supporting Information). The solution was further sonicated to test if the particles could be converted into smaller sizes in the LUV or SUV range. However, DLS yielded a similar result, indicating that the phospholipid analogue does not form stable LUVs but potentially inverted hexagonal phases. Contrarily, (R)-inverted ester showed a sharp phase transition (Figure S5C, Supporting Information). To quantify the ability of sPLA2 to hydrolyze phospholipid analogues, we used light scattering as an indirect measure and HPLC and MALDI-TOF MS for quantitative measurements of hydrolysis. After the addition of the enzyme, changes in light scattering were observed for (R)-inverted ester but not for (R)triple alkyl (Figures S5D and S5E, respectively, Supporting Information). HPLC and MALDI-TOF MS results, on the other hand, showed that sPLA2 is capable of hydrolyzing both phospholipid analogues (Figure 6 and Figure S6, Supporting Information). This is in contrast with previous results, where changes in light scattering were indicative of sPLA2 hydrolysis, which was subsequently confirmed by HPLC.70 The controversy can be explained by the very different behavior of (R)-triple alkyl compared to that of natural substrates (PC, PG). Before the addition of the enzyme, a very complex hexagonal mixture is probably present in the (R)-triple alkyl aggregate solution. After the addition of the enzyme, the hydrolysis products include diacyl phospholipids, which themselves form lipid bilayers. It is therefore expected that the hydrolysis products are also capable of forming larger particles (as observed from the DLS data; Figure S5B, Supporting Information), and no morphology changes would be observed after the addition of sPLA2. Interestingly, in view of previous results, where we showed that short substituents in the sn-1 position can interfere with sPLA2 activity,70 the present results show that sPLA2 tolerates phospholipid analogues with large substituents in the sn-1 position as long as the substituents can reach out of the active site and be embedded back into the lipid layer.
4. CONCLUSIONS To conclude, we have characterized a number of phospholipid analogues and performed a series of MD simulations of sPLA2 phospholipid analogue complexes to investigate how structural
modification in the phospholipids affects their ability to form liposomes and to determine sPLA2 activity toward these lipids. Our results indicate that these substrates are efficiently hydrolyzed and are optimally placed in the binding cleft, allowing free access of water molecules to the active site. Water molecules enter the active site cavity via a hydrophobic groove, where Phe5 (restrained by Ile9) and Tyr51 act as hydrophobic walls, diverting the incoming water molecules toward the active site cavity. The importance of these residues for maintaining enzyme activity has been established by mutational studies.91,9396 Only the phospholipid analogue with a relatively long sn-1 side chain does not form liposomes. This lipid forms aggregates of different shapes with no well-defined sizes due to its relatively small head group to large hydrophobic chain volume ratio.100 Hence, this analogue will not form vesicles and may therefore only be used in combination with other phospholipids for drug carriers. Both head groups, phosphatidylglycerol and phosphatidylcholine, interact with charged residues located in the vicinity of the binding cleft. Head groupGlu55 interaction was most pronounced. However, large fluctuations in these interactions were observed, suggesting that these are not necessarily important for stabilizing substrate binding to the enzyme.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional structural information extracted from the MD simulations and experiments (average distances, Table S1; relative and absolute water counts, Table S2; atom types that are given by the Charmm27 parameter set used for describing (R)-inverted ester, (S)- and (R)-triple alkyl, Figure S1; time evolution of the rmsd for PG, (R)-inverted ester, (R)-triple alkyl, and (S)-triple alkyl, Figure S2; time evolution of selected distances between PClipid head group atoms and protein residues, Figure S3; time evolution of selected distances between PGlipid head group atoms and protein residues, Figure S4; DSC scan for (R)-triple alkyl, Figures S5A; DLS scan for (R)triple alkyl, Figure S5B; DSC scan for (R)-inverted ester, Figure S5C; light scattering measurements for (R)-inverted ester and (R)triple alkyl, Figure S5D and S5E, respectively; and MALDI-TOF 6859
dx.doi.org/10.1021/jp112137b |J. Phys. Chem. B 2011, 115, 6853–6861
The Journal of Physical Chemistry B data for (R)-inverted ester, Figure S6). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (þ45) 45252486. Fax: (þ45) 45883136. E-mail:
[email protected].. Present Addresses ^
Novo Nordisk A/S, Protein & Peptide Chemistry 3, Novo Denmark. Nordisk Park, DK-2760 Maløv,
’ ACKNOWLEDGMENT G.H.P. acknowledges financial support from the Danish National Research Foundation via a grant to the MEMPHYS-Center for Biomembrane Physics. Simulations were performed at the Danish Center for Scientific Computing at the University of Southern Denmark. ’ REFERENCES (1) Six, D. A.; Dennis, E. A. Biochim. Biochim. Acta 2000, 1488, 1–19. (2) White, S. P.; Scott, D. L.; Otwinowski, Z.; Gelb, M. H.; Sigler, P. B. Science 1990, 250, 1560–1563. (3) Scott, D. L.; Otwinowski, Z.; Gelb, M. H.; Sigler, P. B. Science 1990, 250, 1563–1566. (4) Kudo, I.; Murakami, M. Prostaglandins Other Lipid Mediators 2002, 6869, 3–58. (5) Valentin, E.; Lambeau, G. Biochim. Biochim. Acta 2000, 1488, 59–70. (6) Dennis, E. A. J. Biol. Chem. 1994, 269, 13057–13060. (7) Murakami, M. Biol. Pharm. Bull. 2004, 27, 1179–1182. (8) Diaz, B. L.; Arm, J. P. Essent. Fatty Acids 2002, 69, 87–97. (9) Singer, A. G.; Ghomashchi, F.; Le Calvez, C.; Bollinger, J.; Bezzine, S.; Rouault, M.; Sadilek, M.; Nguyen, E.; Lazdunski, M.; Lambeau, G.; Gelb, M. H. J. Biol. Chem. 2002, 277, 48535–48549. (10) Scott, D. L.; White, S.; Otwinowski, Z.; Yuan, W.; Gelb, M. H.; Sigler, P. B. Science 1990, 250, 1541–1546. (11) Scott, D. L.; Sigler, P. B. Adv. Protein Chem. 1994, 45, 53–88. (12) Poi, M. J.; Tomaszewski, J. W.; Yuan, C.; Dunlap, C. A.; Andersen, N. H.; Gelb, M. H.; Tsai, M.-D. J. Mol. Biol. 2003, 329, 997–1009. (13) Blow, D. M.; Birktoft, J. J.; Hartley, B. S. Nature 1969, 221, 337–340. (14) Janssen, M. J.; van de Wiel, W. A.; Beiboer, S. H.; van Kampen, M. D.; Verheij, H. M.; Slotboom, A. J.; Egmond, M. R. Protein Eng. 1999, 12, 497–503. (15) Sekar, K.; Yu, B.-Z.; Rogers, J.; Lutton, J.; Liu, X.; Chen, X.; Tsai, M.-D.; Jain, M. K.; Sundaralingam, M. Biochemistry 1997, 36, 3104–3114. (16) Pickard, R. T.; Chiou, X. G.; Strifler, B. A.; DeFelippis, M. R.; Hyslop, P. A.; Tebbe, A. L.; Yee, Y. K.; Reynolds, L. J.; Dennis, E. A.; Kramer, R. M.; Sharp, J. D. J. Biol. Chem. 1996, 271, 19225–19231. (17) Kramer, R. M.; Hession, C.; Johansen, B.; Hayes, G.; McGray, P.; Pingchang, C. E.; Tizard, R.; Blake, P. R. J. Biol. Chem. 1989, 264, 5768–5775. (18) Bonventre, J. V. J. Am. Soc. Nephrol. 1992, 3, 128–150. (19) Dessen, A. Biochim. Biochim. Acta 2000, 1488, 40–47. (20) Berg, O. G.; Gelb, M. H.; Tsai, M.-D.; Jain, M. K. Chem. Rev. 2001, 101, 2613–2653. (21) Tatulian, S. A.; Biltonen, R. L.; Tamm, L. K. J. Mol. Biol. 1997, 268, 809–815. (22) Scott, D. L.; Mandel, A. M.; Sigler, P. B.; Honig, B. Biophys. J. 1994, 67, 493–504. (23) Jain, M. K.; Ranadive, G.; Yu, B.-Z.; Verheij, H. M. Biochemistry 1991, 30, 7330–7340.
ARTICLE
(24) Peters, G. H.; Dahmen-Levison, U.; de Meijere, K.; Brezesinski, G.; Toxvaerd, S.; Svendsen, A.; Kinnunen, P. K. J. Langmuir 2000, 16, 2779–2788. (25) Mouritsen, O. G.; Andresen, T. L.; Halperin, A.; Hansen, P. L.; Jakobsen, A. F.; Jensen, U. B.; Jensen, M. Ø.; Jørgensen, K.; Kaasgaard, T.; Leidy, C.; Simonsen, A. C.; Peters, G. H.; Weiss, M. J. Phys.: Condens. Matter 2006, 18, 1–12. (26) Leidy, C.; Linderoth, L.; Andresen, T. L.; Mouritsen, O. G.; Jørgensen, K.; Peters, G. H. Biophys. J. 2006, 90, 3165–3175. (27) Leidy, C.; Mouritsen, O. G.; Jørgensen, K.; Peters, G. H. Biophys. J. 2004, 87, 408–418. (28) Gadd, M. E.; Biltonen, R. L. Biochemistry 2000, 39, 9623–9631. (29) Pan, Y. H.; Yu, B.-Z.; Singer, A. G.; Ghomashchi, F.; Lambeau, G.; Gelb, M. H.; Jain, M. K.; Bahnson, B. J. J. Biol. Chem. 2002, 277, 29086–29093. (30) Bahnson, B. J. Arch. Biochem. Biophys. 2005, 433, 96–106. (31) Canaan, S.; Nielsen, R.; Ghomashchi, F.; Robinson, B. H.; Gelb, M. H. J. Biol. Chem. 2002, 277, 30984–30990. (32) Han, S. K.; Yoon, E. T.; Scott, D. L.; Sigler, P. B.; Cho, W. J. Biol. Chem. 1997, 272, 3573–3582. (33) Yu, B.-Z.; Poi, M. J.; Ramagopal, U. A.; Jain, R.; Ramakumar, S.; Berg, O. G.; Tsai, M. D.; Sekar, K.; Jain, M. K. Biochemistry 2000, 39, 12312–12323. (34) Murakami, M.; Koduri, R. S.; Enomoto, A.; Shimbara, S.; Seki, M.; Yoshihara, K.; Singer, A.; Valentin, E.; Ghomashchi, F.; Lambeau, G.; Gelb, M. H.; Kudo, I. J. Biol. Chem. 2001, 276, 10083–10096. (35) Kim, Y. J.; Kim, K. P.; Han, S. K.; Munoz, N. M.; Zhu, X.; Sano, H.; Leff, A. R.; Cho, W. Biol. Chem. 2002, 277, 36479–36488. (36) Kudo, I.; Murakami, M.; Hara, S.; Inoue, K. Biochim. Biochim. Acta 1993, 1170, 217–231. (37) Andersen, S.; Sjursen, W.; Laegreid, A.; Volden, G.; Johansen, B. Inflammation 1994, 18, 1–12. (38) Chen, Y.; Dennis, E. A. Biochim. Biophys. Acta 1998, 1394, 57–64. (39) Degousee, N.; Ghomashchi, F.; Stefanski, E.; Singer, A.; Smart, B. P.; Borregaard, N. J. Biol. Chem. 2002, 277, 5061–5073. (40) Wolbink, G. J.; Bossink, A. W.; Groeneveld, A. B.; de Groot, M. C.; Thijs, L. G.; Hack, C. E. J. Infect. Dis. 1998, 177, 81–87. (41) Green, J. A.; Smith, G. M.; Buchta, R.; Lee, R.; Ho, K. Y.; Rajkovic, I. A. Inflammation 1991, 15, 355–367. (42) Vadas, P.; Keystone, J.; Stefanski, E.; Scott, K.; Pruzanski, W. Infect. Immun. 1992, 60, 3928–3931. (43) Aarsman, A. J.; Neys, F. W.; var der Helm, H. A.; Kuypers, F. A.; van den Bosch, H. Biochim. Biophys. Acta 2000, 1502, 257–263. (44) Styles, L. A.; Schalkwijk, C. G.; Aarsman, A. J.; Vichinsky, E. P.; Lubin, B. H.; Kuypers, F. A. Blood 1996, 87, 2573–2578. (45) Glomset, J. A. Curr. Opin. Struct. Biol. 1999, 9, 425–427. (46) Abe, T.; Sakamoto, K.; Kamohara, H.; Hirano, Y.; Kuwahara, N.; Ogawa, M. Int. Cong. Ser. 2003, 1255, 351–360. (47) Abe, T.; Sakamoto, K.; Kamohara, H.; Hirano, Y.; Kuwahara, N.; Ogawa, M. Int. J. Cancer 1997, 74, 245–250. (48) Sved, P.; Scott, K. F.; McLeod, D.; King, N. J. C.; Singh, J.; Tsatralis, T.; Nikolov, B.; Boulas, J.; Nallan, L.; Gelb, M. H.; Sajinovic, M.; Graham, G. G.; Russell, P. J.; Dong, Q. Cancer Res. 2004, 64, 6934–6940. (49) Jiang, J.; Neubauer, B. L.; Graff, J. R.; Chedid, M.; Thomas, J. E.; Roehm, N. W.; Zhang, S.; Eckert, G. J.; Koch, M. O.; Eble, J. N.; Cheng, L. Am. J. Pathol. 2002, 160, 667–671. (50) Ilsley, J. N. M.; Nakanishi, M.; Flynn, C.; Belinsky, G. S.; De Guise, S.; Adib, J. N.; Dobrowsky, R. T.; Bonventre, J. V.; Rosenberg, D. W. Cancer Res. 2005, 65, 2636–2643. (51) Edhemovic, I.; Snoj, M.; Kljun, A.; Golouh, R. Eur. J. Surg. Oncol. 2001, 27, 545–548. (52) Yamashita, S.; Yamashita, J.; Ogawa, M. Br. J. Cancer 1994, 69, 1166–1170. (53) Kiyohara, H.; Egami, H.; Kurizaki, T.; Murata, K.; Ohmachi, H.; Akagi, J.; Ohshima, S.; Yamamoto, S.; Shibata, Y.; Ogawa, M. Int. Cong. Ser. 2003, 1255, 375–379. (54) Hanada, K.; Kinoshita, E.; Itoh, M.; Hirata, M.; Kajiyama, G.; Sugiyama, M. FEBS Lett. 1995, 373, 85–87. 6860
dx.doi.org/10.1021/jp112137b |J. Phys. Chem. B 2011, 115, 6853–6861
The Journal of Physical Chemistry B (55) Andresen, T. L.; Jensen, S. S.; Jørgensen, K. Prog. Lipid Res. 2005, 44, 68–97. (56) Andresen, T. L.; Jensen, S. S.; Kaasgaard, T.; Jørgensen, K. Curr. Drug Delivery 2005, 2, 353–362. (57) Davidsen, J.; Jørgensen, K.; Andresen, T. L.; Mouritsen, O. G. Biochim. Biophys. Acta 2003, 1609, 95–101. (58) Mihelich, E. D.; Schevitz, R. W. Biochim. Biophys. Acta 1999, 1441, 223–228. (59) Peters, G. H.; Møller, M. S.; Jørgensen, K.; R€onnholm, P.; Mikkelsen, M.; Andresen, T. L. J. Am. Chem. Soc. 2007, 129, 5451–5461. (60) Linderoth, L.; Peters, G. H.; Jørgensen, K.; Madsen, R.; Andresen, T. L. Chem. Phys. Lipids 2006, 146, 54–66. (61) Singh, U. C.; Kollman, P. A. J. Comput. Chem. 1986, 7, 718–730. (62) Burton, N. A.; Harrison, M. J.; Hart, J. C.; Hillier, I. H.; Sheppard, D. W. Faraday Discuss. 1998, 110, 463–475. (63) Rothlisberger, U.; Carloni, P.; Doclo, K.; Parrinello, M. J. Biol. Inorg. Chem. 2000, 5, 236–250. (64) Shokhen, M.; Albeck, A. Proteins 2000, 40, 154–167. (65) Bash, P. A.; Field, M. J.; Davenport, R. C.; Petsko, G. A.; Ringe, D.; Karplus, M. Biochemistry 1991, 30, 5826–5832. (66) Åqvist, J.; Warshel, A. Chem. Rev. 1993, 93, 2523–2544. (67) Feierberg, I.; Cameron, A. D.; Åqvist, J. FEBS Lett. 1999, 453, 90–94. (68) Villa, J.; Warshel, A. J. Phys. Chem. B 2001, 105, 7887–7907. (69) Warshel, A. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 98–107. (70) Linderoth, L.; Andresen, T. L.; Jørgensen, K.; Madsen, R.; Peters, G. H. Biophys. J. 2008, 94, 14–26. (71) Linderoth, L. Enzyme triggered drug delivery. Ph.D. dissertation, Department of Chemistry, Technical University of Denmark, 2008. (72) Peters, G. H.; Iversen, L. F.; Andersen, H. S.; Møller, N. P. H.; Olsen, O. H. Biochemistry 2004, 43, 8418–8428. (73) Peters, G. H.; Iversen, L. F.; Branner, S.; Andersen, H. S.; Mortensen, S. B.; Olsen, O. H.; Moller, K. B.; Moller, N. P. J. Biol. Chem. 2000, 275, 18201–18209. (74) Linderoth, L.; Fristrup, P.; Hansen, M.; Melander, F.; Madsen, R.; Andresen, T. L.; Peters, G. H. J. Am. Chem. Soc. 2009, 131, 12193–12200. (75) Linderoth, L.; Peters, G. H.; Madsen, R.; Andresen, T. L. Angew. Chem. 2009, 121, 1855–1858. (76) Pedersen, P. J.; Christensen, M. S.; Ruysschaert, T.; Linderoth, L.; Andresen, T. L.; Melander, F.; Mouritsen, O. G.; Madsen, R.; Clausen, M. H. J. Med. Chem. 2009, 52, 3408–3415. (77) Landau, M.; Mayrose, I.; Rosenberg, Y.; Glaser, F.; Martz, E.; Pupko, T. Nucleic Acids Res. 2005, 33, W299–W302. (78) Glaser, F.; Pupko, T.; Paz, I.; Bell, R. E.; Bechor, D.; Martz, E.; Ben-Tal, N. Bioinformatics 2003, 19, 163–164. (79) Mayrose, I.; Graur, D.; Ben-Tal, N.; Pupko, T. Mol. Biol. Evol. 2004, 21, 1781–1791. (80) Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D. J. Nucleic Acids Res. 1997, 25, 3389–3402. (81) Hansford, K. A.; Reid, R. C.; Clark, C. I.; Tyndall, J. D. A.; Whitehouse, M. W.; Guthrie, T.; McGeary, R. P.; Schafer, K.; Martin, J. L.; Fairlie, D. P. ChemBioChem 2003, 4, 181–185. (82) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J.; Meyer, E. E.; Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M. J. Mol. Biol. 1977, 112, 535–542. (83) Grubm€uller, H.; Groll, V. Solvate: a program to create atomic solvent models; Unversity of Munich: Germany, 1996. (84) Kale, L.; Skeel, R.; Bhandarkar, M.; Brunner, R.; Gursoy, A.; Krawetz, N.; Phillips, J.; Shinozaki, A.; Varadarajan, K.; Schulten, K. J. Comput. Phys. 1999, 151, 283–312. (85) Jorgensen, W. L.; Chandrasekhar, J.; Medura, J. D.; Impey, R. W.; Klein, M. L. J. Chem. Phys. 1983, 79, 926–935. (86) Feller, S. E.; Zhang, Y.; Pastor, R. W.; Brooks, B. R. J. Chem. Phys. 1995, 103, 4613–4621. (87) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 10089–10092. (88) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577–8593.
ARTICLE
(89) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graph. 1996, 14, 33–38. (90) Garcia, A. E.; Hummer, G. Proteins 2000, 38, 261–272. (91) Liu, X.; Zhu, H.; Huang, B.; Rogers, J.; Yu, B. Z.; Kumar, A.; Jain, M. K.; Sundaralingam, M.; Tsai, M. D. Biochemistry 1995, 34, 7322–7334. (92) Yuan, C.; Tsai, M.-D. Biochim. Biophys. Acta 1999, 1441, 215–222. (93) Dupureur, C. M.; Yu, B.-Z.; Jain, M. K.; Noel, J. P.; Deng, T.; Li, Y.; Byeon, I.-J. L.; Tsai, M.-D. Biochemistry 1992, 31, 6402–6413. (94) Dupureur, C. M.; Deng, T.; Kwak, J. G.; Noel, J. P.; Tsai, M.-D. J. Am. Chem. Soc. 1990, 112, 7074–7076. (95) Kumar, A.; Sekharudu, C.; Ramakrishnan, B.; Dupureur, C. M.; Zhu, H.; Tsai, M. D.; Sundaralingam, M. Protein Sci. 1994, 3, 2082–2088. (96) Dupureur, C. M.; Yu, B. Z.; Mamone, J. A.; Jain, M. K.; Tsai, M. D. Biochemistry 1992, 31, 10576–10583. (97) Scott, D. L.; White, S. P.; Browning, J. L.; Rosa, J. J.; Gelb, M. H.; Sigler, P. B. Science 1991, 254, 1007–1010. (98) Leidy, C.; Kaasgaard, T.; Mouritsen, O. G.; Jørgensen, K.; Peters, G. H. Recent Res. Dev. Biophys. 2004, 3, 163–185. (99) Christensen, M. S.; Pedersen, P. J.; Andresen, T. L.; Madsen, R.; Clausen, M. H. Eur. J. Org. Chem. 2010, 2010, 719–724. (100) Mouritsen, O. G. Life — As a Matter of Fat. The Emerging Science of Lipidomics; Springer-Verlag: Berlin, Germany, 2004.
6861
dx.doi.org/10.1021/jp112137b |J. Phys. Chem. B 2011, 115, 6853–6861
