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pK values of titrable amino acids at the water/membrane interface Vitor H. Teixeira, Diogo Vila-Viçosa, Pedro B. P. S. Reis, and Miguel Machuqueiro J. Chem. Theory Comput., Just Accepted Manuscript • DOI: 10.1021/acs.jctc.5b01114 • Publication Date (Web): 10 Feb 2016 Downloaded from http://pubs.acs.org on February 12, 2016
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pKa values of titrable amino acids at the water/membrane interface Vitor H. Teixeira, Diogo Vila-Vi¸cosa, Pedro B. P. S. Reis, and Miguel Machuqueiro∗ Centro de Qu´ımica e Bioqu´ımica, Departamento de Qu´ımica e Bioqu´ımica, Faculdade de Ciˆencias, Universidade de Lisboa, 1749-016 Lisboa, Portugal E-mail: [email protected] Phone: +351-21-7500112. Fax: +351-21-7500088 Abstract Peptides and proteins protonation equilibrium is strongly influenced by its surrounding media. Remarkably until now, there are no quantitative and systematic studies reporting the pKa shifts in the common titrable amino acids upon lipid membrane insertion. Here, we applied our recently developed CpHMD-L method to calculate the pKa values of titrable amino acid residues incorporated in Ala-based pentapeptides at the water/membrane interface. We observed that membrane insertion leads to desolvation and a clear stabilization of the neutral forms, and we quantified the increases/decreases of the pKa values in the anionic/cationic residues along the membrane normal. This work highlights the importance of properly modeling the protonation equilibra of peptides and proteins interacting with membranes using molecular dynamics simulations.
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Introduction pH is a crucial physicochemical property that affects most biomolecules. Changes in protonation equilibrium of susceptible sites will modify the electrostatic environment and, consequently, have an effect on the molecular structure, stability and catalysis. 1–5 The proton avidity of the titrable sites (i.e. their pKa ) is strongly influenced by the level of solvent exposure (buried or exposed) and by the electrostatic interactions with neighbor polar groups. As an example, when a protein unfolds, the pKa values of several amino acids are significantly shifted. 6,7 The pKa values of the typical titrable amino acids can also be influenced by changes in the solvent mixture 8 or the insertion into a lipid bilayer. 9–21 The protonation behavior of peptides interacting with membranes has also attracted large interest in the context of cell-penetrating peptides 22 and pH-dependent membrane insertion peptides. 9–12 In fact, it was recently showed that a faster membrane permeation is obtained with a positive tryptophan, challenging the electrostatics-based rule where the neutral form should be the most stable. 23 Membranes are very complex systems, creating large difficulties to most experimental techniques in successfully studying protonation of biomolecules in this environment. In some cases, it is possible to follow the protonation of certain groups, because they are coupled to other events which are easier to measure, which is the case of the pHLIP peptide. 9–12 In this case, an insertion pK is measured, as an estimation of the pKa of the group at the last moment of membrane insertion where it can still exchange protons with the solvent. In light of these limitations, computational methodologies appear as a valuable tool to study the pH effect in lipid bilayer systems. 14,17,18,24–27 A seminal study on the energetics of peptides interacting with membranes was carried by McLaughlin and co–workers, 28 where they calculated the binding electrostatic energy of a basic peptide interacting with an explicit acidic lipid bilayer. More recent computational studies, aimed to understand how peptides interact, insert and partition into lipid bilayers 15,29–37 and how pH can influence these phenomena. 14,17 For pH sensitive peptides, 2
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like kyotorphin, it was necessary to consider the protonation equilibrium to calculate its conformational preferences in the water/membrane interface. 14,17 Recently, we have extended the stochastic titration constant-pH molecular dynamics method (CpHMD) 38,39 to include lipid bilayers in these simulations (CpHMD-L), where lipids are also able to titrate, if necessary. 27 This was accomplished by simulating small patches of phospholipids and considering periodic boundary conditions in both molecular mechanics (MM) and Poisson–Boltzmann (PB) calculations. 40 Our CpHMD-L method is currently a state-of-the-art methodology to study pH effects in lipid bilayers and in proteins / peptides interacting with membranes. Alanine-based pentapeptides are simple molecules but very useful in the field of computational biophysics. These pentapeptides (Ala2 – X –Ala2 , where X is a pH titrating residue) were initially designed in Pace’s group 41,42 to study the effect of the protein environment on the protonation behavior of titrable residues. The experimentally measured pKa values of these peptides were already used to calibrate the pKa values of the model compounds needed in PB calculations of CpHMD simulations. 8,43,44 Also, the relatively small size of the pentapeptides renders them ideal systems in the study of how lipid membranes can influence the pKa values of pH susceptible amino acid residues. The main objective of this work is a comprehensive study of how a membrane environment can shift the pKa values of common pH-sensitive amino acids (Asp, Glu, His, Lys, Cys, Tyr, and the N- and C-termini). For this, we used our recently developed CpHMD-L methodology with a DMPC membrane and the model Ala-based pentapeptides, already well characterized in water by Pace and co–workers. 41,42 With this approach, we intend to capture the coupling between conformation / configuration / insertion and protonation at the membrane interface, and focus on the pKa changes of the peptides along the membrane normal. We were also interested in identifying pH dependent conformational patterns, lipid region preferences, and kinetic traps. As far as we know, this is the first systematic study aiming to estimate the pKa values of amino acid residues when a peptide is inserting into a membrane.
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Methods We used the recently developed CpHMD-L method 27,40 to simulate eight pentapeptides with Ala in all residue positions, with the exception in the middle which was changed to Asp, Cys, Glu, His, Tyr or Lys. All N- and C-termini were capped with an acetyl or amino groups, respectively. When these termini were to be titrated, they were uncapped in an Ala pentapeptide. For each system, we simulated three pH values with three (aqueous) or five (membrane) replicates. The simulations were 30 and 100 ns long in water and membrane, respectively. The GROMACS package version 4.0.7 45,46 was used in all MM/MD simulations with the GROMOS 54A7 force field 47,48 which already includes the corrections for the DMPC molecule. 49 The atomic partial charges were taken from Teixeira et al. 40 Detailed simulation settings, protocols, and energy landscapes are provided in Supporting Information.
Results and discussion The CpHMD-L method 27 uses the DelPhi package 50,51 to solve the PB equation. The model compounds pKa values previously reported 8,44 using the CpHMD methodology were calibrated using MEAD. 52 Therefore, to take advantage of the new developed methodology to study protein–lipid interactions, we decided to reparameterize the model compound pKa values of the pentapeptides studied by Pace and coworkers 41,42 (Table S1). The observed shifts are small, as expected since the same theoretical model is used in both approaches. However, there are some inherent differences between DelPhi and MEAD which resulted in the observed pKa shifts. These can be related to numerical errors regarding differences in grid positions, surface algorithms, and convergence criteria. Therefore, in this work, we adopted the model compound pKa values calibrated using DelPhi. In the CpHMD-L simulations, the titrable groups are allowed to change their protonation states along the simulation. Therefore, it is possible to evaluate which protonation states are preferred at a given pH value when the peptide is interacting or even inserting into 4
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Figure 1: Residue pKa values along the membrane normal. Negative insertion values correspond to deeper membrane insertions, while positive correspond to more shallow locations. The insertion values were measured between the titrable group and the phosphate from the nearest lipid (see Methods for details). The aqueous bulk pKa values of the pentapeptides are shown on top for comparison. Ctr and Ntr correspond to the C- and N-terminus, respectively. The two horizontal lines at ∼1 ˚ A and ∼–6 ˚ A correspond to the average positions of nd the choline nitrogens and the 2 carbon atoms of the acyl chains, respectively.
the membrane. From simulations at three different pH values and using a Henderson– Hasselbalch fit, we can obtain the pKa values of each residue. In the membrane simulations, we can separate our system in slices along the membrane normal where the pKa values of titrable group can be fitted at different membrane insertion depths (Figure 1). In all cases, the pKa values away from the membranes (∼10 ˚ A) are lower than their bulk references, which can be explained by the presence of a positive electrostatic potential generated by the nearby choline groups. This effect induces a lowering of the pKa values in both anionic and cationic residues. The membrane insertion leads to increasing desolvation of the titrable group, resulting in an stabilization of their neutral forms. As a consequence, we observe a significant increase of the pKa values of the anionic groups, while the cationic ones undergo an opposite variation. We were unable to measure the pKa profile for all residues to the same insertion depth, however, this does not mean the pentapeptides are inserting unequally. This is due to a limitation in our pKa calculation procedure, where we need enough proton exchange sampling
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to do the fit. Therefore, in some residues (C-ter, Asp, Cys, and Tyr) the peptides are able to insert quite deep in the membrane, but because they do not ionize in those regions, we are not able to estimate their pKa values. We would probably need to run these simulations at much higher (anionic groups) or lower (cationic groups) pH values and for longer simulation times. The anionic carboxylic acids pKa shifts range from ∼1.5 (C-ter) to ∼3.0 pH units (Glu) upon membrane internalization. The large increase in their pKa values is associated with desolvation, favoring the neutral forms, while the ionized carboxylates are also destabilized in the phosphate and ester group regions. These pKa shifts have already been observed in pH-triggered fusion proteins such as the hemagglutinins from influenza virus 53,54 and in pH-dependent membrane insertion peptides, like pHLIP. 9–11 In the case of pHLIP peptide, with two Asp residues in the transmembrane region, the authors report insertion pK values as high as 6.2. 12 The other two anionic residues, Tyr and Cys, are even more sensitive to membrane insertion and their pKa values are shifted ∼3.5 pH units. The same arguments given for carboxylic acids are valid and the slightly larger shifts are probably related with the fact that, in this case, when ionized, the negative charge is concentrated in only one atom, creating a larger local dipole compared with the charge distribution present in the carboxylate group. The N-terminus is able to titrate in very deep positions of the membrane (∼6 ˚ A below the nearest phosphate group) and, consequently, we measured a significant pKa shift (∼3 pH units). This was possible because, as a terminal group, it can reach far deeper in the membrane and its positive form may be stabilized by oxygen atoms from neighbor phosphate and ester groups. In the case of Lysine, the protonable group is located at the center of the pentapeptide where it probably has more conformational restrictions. This reduces the sampling in deeper regions of the membrane and consequently makes it difficult to obtain the large shift values observed for N-terminus. Histidine showed a smaller pKa shift (∼1.0 pH units) probably due to the fact that, upon ionization, the positive charge is distributed
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over the whole imidazole, which becomes easier to stabilize by the neighboring groups. The small increase in the pKa values around −5 ˚ A insertion can be attributed to the previously mentioned cation stabilization in the region between the phosphate and ester groups. In order to investigate the conformational space of the pentapeptide interacting with the membrane, we followed the radius of gyration at different insertion regions (see Supporting Information). In most cases, we observe large continuous energy basins outside and in a shallow region of the membrane. Also, in these regions, there is a large overlap between replicas (data not shown). Altogether, these results indicate that most pentapeptides are able to sample their conformational space without many restrictions or kinetic traps, however, some noteworthy cases occurred (see examples in Figures S1–2). It should be mentioned that these special cases have no significant influence on the final reported pKa values. A pentapeptide is not large enough to present significant secondary structure. However, we observed a high content of 310 helix content in one replicate of the Glu pentapeptide simulations (top panel of Figure 2). These more helical conformations favor the peptide insertion into the membrane (middle panel of Figure 2) coupled with a higher degree of protonation on the Glu side chain (0.94 in replicate 1 compared with 0.60 in replicate 2 at pH 4.0). The membrane role is pivotal, since in water, the hydrogen bonds in the helical structures are not stable enough (data not shown). The bottom panel in Figure 2 shows Glu pentapeptide in a helical conformation interacting with the ester groups region of the neighbor lipids. These inserted helical conformations were also captured in a separate basin of the energy landscape (central panel of Figure S1). The N-terminus uncapped Ala pentapeptide was able to stabilize its neutral form by forming an intramolecular hydrogen bond. This locked conformation was stable for long periods of our simulation (top panel of Figure 3) and also facilitates a deeper insertion of the pentapeptide into the membrane (center panel of Figure 3). The hydrogen bond is formed between the lone pair of the neutral primary amine, acting as the acceptor, and the NH group of the second Ala residue, as the donor (bottom panel of Figure 3). Its high
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Figure 2: The ALA2 -GLU-ALA2 pentapeptide is able to form stable 310 helix structures in one of our simulations at pH 4.0 (top panel). Hrx refers to the 310 helix conformation of replicate x, while Crx refers to coil (just the non helical conformations, for simplicity). Two different replicates are shown for comparison between a regular simulation (blue) and the special case (red). Their membrane insertion profiles are shown in the middle panel. The snapshot in the bottom panel was obtained at the simulation time marked with the green circle.
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Figure 3: The N-terminus pentapeptide can form an intramolecular locked conformation. The top panel shows the hydrogen bond distance at pH 7.0. Two different replicates are shown for comparison between a regular simulation (blue) and the special case (red). Their membrane insertion profiles are shown in the middle panel. The snapshot in the bottom panel was obtained at the simulation time marked with the green circle.
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stability was potentiated by insertion and desolvation due to the absence of water molecules to compete for the hydrogen bond. In bulk water simulations, this locked conformation was never observed (data not shown).
Conclusions We applied our recently developed CpHMD-L implementation 27 of the CpHMD method 38,39 to the study of the protonation of Ala-based pentapeptides incorporating all titrable amino acids in the water/membrane interface. The peptides approaching a PC membrane sense the positive charge from the choline groups, which will lower their pKa values. Membrane insertion leads to desolvation and a clear stabilization of the neutral forms. Consequently, the anionic residues have their pKa values increased, while the cationic ones are shifted in the opposite direction. Some residues are able to ionize deep in the membrane which allowed us to measure their pKa values in these regions. The small pentapeptides used 41,42 were also chosen to guarantee a good conformational sampling in our simulations. In overall, this was achieved, even though we were able to identify a few unexpected kinetic traps favored by the membrane environment. It was the case of the N-terminus locked conformation exhibiting an internal hydrogen bond between its neutral form and the NH group from the second residue. Also, we observed an abnormally high content of 310 helix in a Glu pentapeptide simulation, which could only be stabilized in deep regions of the lipid bilayer. To obtain more complete pKa profiles and capture more complex conformational behaviors of the studied pentapeptides, we will need to run a more extensive sampling and cover more pH values. Nevertheless, our results completely capture their pKa trends and reveal the molecular bases of the interactions between the titrating groups and the lipid moiety. Furthermore, our results emphasize the necessity of taking in consideration the protonation equilibria in membrane simulations, and that choosing a fixed protonation state can lead to
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biased and erroneous results.
Acknowledgement We thank Maria J. Calhorda, Ant´onio M. Baptista and Rafael Nunes for fruitful discussions. We acknowledge financial support from Funda¸c˜ao para a Ciˆencia e a Tecnologia through grant SFRH/BD/81017/2011, and projects PTDC/QUI-BIQ/113721/2009, UID/MULTI/00612/2013, and PTDC/QEQCOM/5904/2014.
Supporting Information Available Detailed simulation settings, protocols, and energy landscapes. This material is available free of charge via the Internet at http://pubs.acs.org/.
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pK values of titrable amino acids at the water/membrane interface Vitor H. Teixeira, Diogo Vila-Viçosa, Pedro B. P. S. Reis, and Miguel Machuqueiro J. Chem. Theory Comput., Just Accepted Manuscript • DOI: 10.1021/acs.jctc.5b01114 • Publication Date (Web): 10 Feb 2016 Downloaded from http://pubs.acs.org on February 12, 2016
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pKa values of titrable amino acids at the water/membrane interface Vitor H. Teixeira, Diogo Vila-Vi¸cosa, Pedro B. P. S. Reis, and Miguel Machuqueiro∗ Centro de Qu´ımica e Bioqu´ımica, Departamento de Qu´ımica e Bioqu´ımica, Faculdade de Ciˆencias, Universidade de Lisboa, 1749-016 Lisboa, Portugal E-mail: [email protected] Phone: +351-21-7500112. Fax: +351-21-7500088 Abstract Peptides and proteins protonation equilibrium is strongly influenced by its surrounding media. Remarkably until now, there are no quantitative and systematic studies reporting the pKa shifts in the common titrable amino acids upon lipid membrane insertion. Here, we applied our recently developed CpHMD-L method to calculate the pKa values of titrable amino acid residues incorporated in Ala-based pentapeptides at the water/membrane interface. We observed that membrane insertion leads to desolvation and a clear stabilization of the neutral forms, and we quantified the increases/decreases of the pKa values in the anionic/cationic residues along the membrane normal. This work highlights the importance of properly modeling the protonation equilibra of peptides and proteins interacting with membranes using molecular dynamics simulations.
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Introduction pH is a crucial physicochemical property that affects most biomolecules. Changes in protonation equilibrium of susceptible sites will modify the electrostatic environment and, consequently, have an effect on the molecular structure, stability and catalysis. 1–5 The proton avidity of the titrable sites (i.e. their pKa ) is strongly influenced by the level of solvent exposure (buried or exposed) and by the electrostatic interactions with neighbor polar groups. As an example, when a protein unfolds, the pKa values of several amino acids are significantly shifted. 6,7 The pKa values of the typical titrable amino acids can also be influenced by changes in the solvent mixture 8 or the insertion into a lipid bilayer. 9–21 The protonation behavior of peptides interacting with membranes has also attracted large interest in the context of cell-penetrating peptides 22 and pH-dependent membrane insertion peptides. 9–12 In fact, it was recently showed that a faster membrane permeation is obtained with a positive tryptophan, challenging the electrostatics-based rule where the neutral form should be the most stable. 23 Membranes are very complex systems, creating large difficulties to most experimental techniques in successfully studying protonation of biomolecules in this environment. In some cases, it is possible to follow the protonation of certain groups, because they are coupled to other events which are easier to measure, which is the case of the pHLIP peptide. 9–12 In this case, an insertion pK is measured, as an estimation of the pKa of the group at the last moment of membrane insertion where it can still exchange protons with the solvent. In light of these limitations, computational methodologies appear as a valuable tool to study the pH effect in lipid bilayer systems. 14,17,18,24–27 A seminal study on the energetics of peptides interacting with membranes was carried by McLaughlin and co–workers, 28 where they calculated the binding electrostatic energy of a basic peptide interacting with an explicit acidic lipid bilayer. More recent computational studies, aimed to understand how peptides interact, insert and partition into lipid bilayers 15,29–37 and how pH can influence these phenomena. 14,17 For pH sensitive peptides, 2
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like kyotorphin, it was necessary to consider the protonation equilibrium to calculate its conformational preferences in the water/membrane interface. 14,17 Recently, we have extended the stochastic titration constant-pH molecular dynamics method (CpHMD) 38,39 to include lipid bilayers in these simulations (CpHMD-L), where lipids are also able to titrate, if necessary. 27 This was accomplished by simulating small patches of phospholipids and considering periodic boundary conditions in both molecular mechanics (MM) and Poisson–Boltzmann (PB) calculations. 40 Our CpHMD-L method is currently a state-of-the-art methodology to study pH effects in lipid bilayers and in proteins / peptides interacting with membranes. Alanine-based pentapeptides are simple molecules but very useful in the field of computational biophysics. These pentapeptides (Ala2 – X –Ala2 , where X is a pH titrating residue) were initially designed in Pace’s group 41,42 to study the effect of the protein environment on the protonation behavior of titrable residues. The experimentally measured pKa values of these peptides were already used to calibrate the pKa values of the model compounds needed in PB calculations of CpHMD simulations. 8,43,44 Also, the relatively small size of the pentapeptides renders them ideal systems in the study of how lipid membranes can influence the pKa values of pH susceptible amino acid residues. The main objective of this work is a comprehensive study of how a membrane environment can shift the pKa values of common pH-sensitive amino acids (Asp, Glu, His, Lys, Cys, Tyr, and the N- and C-termini). For this, we used our recently developed CpHMD-L methodology with a DMPC membrane and the model Ala-based pentapeptides, already well characterized in water by Pace and co–workers. 41,42 With this approach, we intend to capture the coupling between conformation / configuration / insertion and protonation at the membrane interface, and focus on the pKa changes of the peptides along the membrane normal. We were also interested in identifying pH dependent conformational patterns, lipid region preferences, and kinetic traps. As far as we know, this is the first systematic study aiming to estimate the pKa values of amino acid residues when a peptide is inserting into a membrane.
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Methods We used the recently developed CpHMD-L method 27,40 to simulate eight pentapeptides with Ala in all residue positions, with the exception in the middle which was changed to Asp, Cys, Glu, His, Tyr or Lys. All N- and C-termini were capped with an acetyl or amino groups, respectively. When these termini were to be titrated, they were uncapped in an Ala pentapeptide. For each system, we simulated three pH values with three (aqueous) or five (membrane) replicates. The simulations were 30 and 100 ns long in water and membrane, respectively. The GROMACS package version 4.0.7 45,46 was used in all MM/MD simulations with the GROMOS 54A7 force field 47,48 which already includes the corrections for the DMPC molecule. 49 The atomic partial charges were taken from Teixeira et al. 40 Detailed simulation settings, protocols, and energy landscapes are provided in Supporting Information.
Results and discussion The CpHMD-L method 27 uses the DelPhi package 50,51 to solve the PB equation. The model compounds pKa values previously reported 8,44 using the CpHMD methodology were calibrated using MEAD. 52 Therefore, to take advantage of the new developed methodology to study protein–lipid interactions, we decided to reparameterize the model compound pKa values of the pentapeptides studied by Pace and coworkers 41,42 (Table S1). The observed shifts are small, as expected since the same theoretical model is used in both approaches. However, there are some inherent differences between DelPhi and MEAD which resulted in the observed pKa shifts. These can be related to numerical errors regarding differences in grid positions, surface algorithms, and convergence criteria. Therefore, in this work, we adopted the model compound pKa values calibrated using DelPhi. In the CpHMD-L simulations, the titrable groups are allowed to change their protonation states along the simulation. Therefore, it is possible to evaluate which protonation states are preferred at a given pH value when the peptide is interacting or even inserting into 4
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Figure 1: Residue pKa values along the membrane normal. Negative insertion values correspond to deeper membrane insertions, while positive correspond to more shallow locations. The insertion values were measured between the titrable group and the phosphate from the nearest lipid (see Methods for details). The aqueous bulk pKa values of the pentapeptides are shown on top for comparison. Ctr and Ntr correspond to the C- and N-terminus, respectively. The two horizontal lines at ∼1 ˚ A and ∼–6 ˚ A correspond to the average positions of nd the choline nitrogens and the 2 carbon atoms of the acyl chains, respectively.
the membrane. From simulations at three different pH values and using a Henderson– Hasselbalch fit, we can obtain the pKa values of each residue. In the membrane simulations, we can separate our system in slices along the membrane normal where the pKa values of titrable group can be fitted at different membrane insertion depths (Figure 1). In all cases, the pKa values away from the membranes (∼10 ˚ A) are lower than their bulk references, which can be explained by the presence of a positive electrostatic potential generated by the nearby choline groups. This effect induces a lowering of the pKa values in both anionic and cationic residues. The membrane insertion leads to increasing desolvation of the titrable group, resulting in an stabilization of their neutral forms. As a consequence, we observe a significant increase of the pKa values of the anionic groups, while the cationic ones undergo an opposite variation. We were unable to measure the pKa profile for all residues to the same insertion depth, however, this does not mean the pentapeptides are inserting unequally. This is due to a limitation in our pKa calculation procedure, where we need enough proton exchange sampling
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to do the fit. Therefore, in some residues (C-ter, Asp, Cys, and Tyr) the peptides are able to insert quite deep in the membrane, but because they do not ionize in those regions, we are not able to estimate their pKa values. We would probably need to run these simulations at much higher (anionic groups) or lower (cationic groups) pH values and for longer simulation times. The anionic carboxylic acids pKa shifts range from ∼1.5 (C-ter) to ∼3.0 pH units (Glu) upon membrane internalization. The large increase in their pKa values is associated with desolvation, favoring the neutral forms, while the ionized carboxylates are also destabilized in the phosphate and ester group regions. These pKa shifts have already been observed in pH-triggered fusion proteins such as the hemagglutinins from influenza virus 53,54 and in pH-dependent membrane insertion peptides, like pHLIP. 9–11 In the case of pHLIP peptide, with two Asp residues in the transmembrane region, the authors report insertion pK values as high as 6.2. 12 The other two anionic residues, Tyr and Cys, are even more sensitive to membrane insertion and their pKa values are shifted ∼3.5 pH units. The same arguments given for carboxylic acids are valid and the slightly larger shifts are probably related with the fact that, in this case, when ionized, the negative charge is concentrated in only one atom, creating a larger local dipole compared with the charge distribution present in the carboxylate group. The N-terminus is able to titrate in very deep positions of the membrane (∼6 ˚ A below the nearest phosphate group) and, consequently, we measured a significant pKa shift (∼3 pH units). This was possible because, as a terminal group, it can reach far deeper in the membrane and its positive form may be stabilized by oxygen atoms from neighbor phosphate and ester groups. In the case of Lysine, the protonable group is located at the center of the pentapeptide where it probably has more conformational restrictions. This reduces the sampling in deeper regions of the membrane and consequently makes it difficult to obtain the large shift values observed for N-terminus. Histidine showed a smaller pKa shift (∼1.0 pH units) probably due to the fact that, upon ionization, the positive charge is distributed
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over the whole imidazole, which becomes easier to stabilize by the neighboring groups. The small increase in the pKa values around −5 ˚ A insertion can be attributed to the previously mentioned cation stabilization in the region between the phosphate and ester groups. In order to investigate the conformational space of the pentapeptide interacting with the membrane, we followed the radius of gyration at different insertion regions (see Supporting Information). In most cases, we observe large continuous energy basins outside and in a shallow region of the membrane. Also, in these regions, there is a large overlap between replicas (data not shown). Altogether, these results indicate that most pentapeptides are able to sample their conformational space without many restrictions or kinetic traps, however, some noteworthy cases occurred (see examples in Figures S1–2). It should be mentioned that these special cases have no significant influence on the final reported pKa values. A pentapeptide is not large enough to present significant secondary structure. However, we observed a high content of 310 helix content in one replicate of the Glu pentapeptide simulations (top panel of Figure 2). These more helical conformations favor the peptide insertion into the membrane (middle panel of Figure 2) coupled with a higher degree of protonation on the Glu side chain (0.94 in replicate 1 compared with 0.60 in replicate 2 at pH 4.0). The membrane role is pivotal, since in water, the hydrogen bonds in the helical structures are not stable enough (data not shown). The bottom panel in Figure 2 shows Glu pentapeptide in a helical conformation interacting with the ester groups region of the neighbor lipids. These inserted helical conformations were also captured in a separate basin of the energy landscape (central panel of Figure S1). The N-terminus uncapped Ala pentapeptide was able to stabilize its neutral form by forming an intramolecular hydrogen bond. This locked conformation was stable for long periods of our simulation (top panel of Figure 3) and also facilitates a deeper insertion of the pentapeptide into the membrane (center panel of Figure 3). The hydrogen bond is formed between the lone pair of the neutral primary amine, acting as the acceptor, and the NH group of the second Ala residue, as the donor (bottom panel of Figure 3). Its high
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Figure 2: The ALA2 -GLU-ALA2 pentapeptide is able to form stable 310 helix structures in one of our simulations at pH 4.0 (top panel). Hrx refers to the 310 helix conformation of replicate x, while Crx refers to coil (just the non helical conformations, for simplicity). Two different replicates are shown for comparison between a regular simulation (blue) and the special case (red). Their membrane insertion profiles are shown in the middle panel. The snapshot in the bottom panel was obtained at the simulation time marked with the green circle.
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Figure 3: The N-terminus pentapeptide can form an intramolecular locked conformation. The top panel shows the hydrogen bond distance at pH 7.0. Two different replicates are shown for comparison between a regular simulation (blue) and the special case (red). Their membrane insertion profiles are shown in the middle panel. The snapshot in the bottom panel was obtained at the simulation time marked with the green circle.
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stability was potentiated by insertion and desolvation due to the absence of water molecules to compete for the hydrogen bond. In bulk water simulations, this locked conformation was never observed (data not shown).
Conclusions We applied our recently developed CpHMD-L implementation 27 of the CpHMD method 38,39 to the study of the protonation of Ala-based pentapeptides incorporating all titrable amino acids in the water/membrane interface. The peptides approaching a PC membrane sense the positive charge from the choline groups, which will lower their pKa values. Membrane insertion leads to desolvation and a clear stabilization of the neutral forms. Consequently, the anionic residues have their pKa values increased, while the cationic ones are shifted in the opposite direction. Some residues are able to ionize deep in the membrane which allowed us to measure their pKa values in these regions. The small pentapeptides used 41,42 were also chosen to guarantee a good conformational sampling in our simulations. In overall, this was achieved, even though we were able to identify a few unexpected kinetic traps favored by the membrane environment. It was the case of the N-terminus locked conformation exhibiting an internal hydrogen bond between its neutral form and the NH group from the second residue. Also, we observed an abnormally high content of 310 helix in a Glu pentapeptide simulation, which could only be stabilized in deep regions of the lipid bilayer. To obtain more complete pKa profiles and capture more complex conformational behaviors of the studied pentapeptides, we will need to run a more extensive sampling and cover more pH values. Nevertheless, our results completely capture their pKa trends and reveal the molecular bases of the interactions between the titrating groups and the lipid moiety. Furthermore, our results emphasize the necessity of taking in consideration the protonation equilibria in membrane simulations, and that choosing a fixed protonation state can lead to
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biased and erroneous results.
Acknowledgement We thank Maria J. Calhorda, Ant´onio M. Baptista and Rafael Nunes for fruitful discussions. We acknowledge financial support from Funda¸c˜ao para a Ciˆencia e a Tecnologia through grant SFRH/BD/81017/2011, and projects PTDC/QUI-BIQ/113721/2009, UID/MULTI/00612/2013, and PTDC/QEQCOM/5904/2014.
Supporting Information Available Detailed simulation settings, protocols, and energy landscapes. This material is available free of charge via the Internet at http://pubs.acs.org/.
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Journal of Chemical Theory and Computation
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Graphical TOC Entry
16
ACS Paragon Plus Environment
Page 16 of 16
