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Molecular Mechanics
Free Energy Calculation of Transmembrane Ion Permeation#Sample
with a Single Reaction Coordinate and Analysis along Transition Path
Xiaoqing Guan, Dong-Qing Wei, and Dan Hu
J. Chem. Theory Comput., Just Accepted Manuscript • DOI: 10.1021/acs.jctc.8b01096 • Publication Date (Web): 15 Jan 2019
Downloaded from http://pubs.acs.org on January 20, 2019

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4 Free Energy Calculation of Transmembrane Ion Permeation ：
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7 Sample with a Single Reaction Coordinate and Analysis along
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9 Transition Path
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12 Xiaoqing Guan,† Dongqing Wei,† and Dan Hu,*,‡
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14 † State Key Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology,
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16 Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China
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18 ‡School of Mathematical Sciences, Institute of Natural Sciences, and MOE-LSC, Shanghai Jiao Tong
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20 University, Shanghai 200240, People’s Republic of China
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23 Abstract
24 A single reaction coordinate is designed to perform umbrella sampling for the water-chain-assist
25
transmembrane permeation processes of ions and highly polar groups. In these permeation processes,
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27 there are three types of configuration changes including deformation of both leaflets of the lipid
28 bilayer, formation of a water chain across the bilayer, and translocation of the ion through the bilayer.
29 Due to the complexity of the permeation process, multiple reaction coordinates and expensive high
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31 dimensional free energy calculations have been used in previous studies. Our single reaction coordinate
32 can be used to obtain sample data of the entire permeation process, which significantly reduces the
33 computational cost in free energy calculations. We show that one-dimensional umbrella sampling along
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35 the new reaction coordinate can be used to obtain continuous sample data roughly along the minimum
36 free energy path (MFEP) on the phase plane of the two components of the new reaction coordinate.
37 Along the MFEP, a one-dimensional bin-segmentation of the sample data is then used to calculate the
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potential of mean force accurately by the newly developed free energy analysis method---weighted
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40 least square analysis method (Welsam). Our work not only proposes a useful reaction coordinate to
41 obtain sample data for water-chain assist transmembrane permeation processes but also establishes a
42 general methodology for one-dimensional free energy calculations with bin-segmentation along the
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44 transition path in multiple dimensional phase space.
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46 Introduction
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48 As the main component of biological membranes, a lipid bilayer provides an effective osmotic barrier
49 for both its contents and molecules in its surrounding environment by its hydrophobic core. This
50 function is crucial for cells and cell organelles to maintain a relatively stable state. The permeation
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rates for molecules with highly polar or charged groups are usually very low due to high energy
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53 barriers for them to pass through the hydrophobic cores of lipid membranes. Nevertheless,
54 transmembrane translocation events of such groups are also involved in many important biological
55 functions. For instance, translocation of antimicrobial peptides (AMP) is a basic step for them to form
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57 transmembrane structures1,2 and translocations of the charged amino acids of many ion channels are
58 key factors to switch the channels between close state and open state.3,4 Detailed information of such
59 translocation processes is crucial for understanding the mechanism of related biological processes and
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3 ultimately sheds light on drug designing and drug delivery.
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5 Despite the importance of many transmembrane translocation processes, there have been very few
6 successful experimental techniques for the study of these processes. Simple measurements of
7 permeation rates5–8 can only provide estimates of the free energy barriers but no information about the
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9 permeation mechanisms.
10 Molecular dynamics (MD) simulation combined with enhanced sampling techniques is a powerful
11 method to obtain detailed information about rare event dynamics. Free energy calculation has been
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widely used in studying transmembrane translocation processes. In the early studies,9–17 a naïve
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14 Reaction Coordinate (RC)—the insertion depth of the solute—is used in umbrella sampling and free
15 energy calculation. Recent studies have shown that this naïve RC leads to statistical and systematic
16 errors due to hidden energy barriers in general, especially for charged and highly polar solutes.16,18–23
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18 Recent reviews have discussed the problems in detail.15,22 The sampling errors would prevent us from
19 obtaining the accurate Potential of Mean Forces (PMFs) and estimating the free energy barriers. Thus,
20 simulations based on this naïve RC cannot be used to precisely describe the entire transition path.
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22 One approach to minimize the errors is to design multiple tailored RCs. With multiple tailored RCs,
23 free energy calculations have been successfully used to obtain correct translocation mechanisms and
24 accurate multi-dimensional PMF profiles.21,24,25 In these studies, both the water-chain assist mechanism
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and the dehydration mechanism are discovered for transmembrane permeation of ions and translocation
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27 of highly polar groups.7,8 The water-chain assist mechanism is further observed in ab-initio simulations
28 of peptide-membrane interactions.2
29 The multi-dimensional PMF profiles can provide useful information for predicting the transition
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31 (permeation) rates and revealing hidden energy barriers in previous studies.13,15,16,19,21–23,25,26
32 Nevertheless, multi-dimensional free energy calculation is inconvenient and expensive in general.
33 What’s more, multiple charged or highly polar groups may be involved in the transmembrane
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35 translocation process of many peptides and membrane proteins, which makes the multi-dimensional
36 free energy calculation very complex. From this point of view, it is important to design a single RC to
37 obtain sample data of transmembrane translocation of a single ion or a single group. Three types of
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configuration changes are involved in the water-chain assist mechanism for ion permeation, including
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40 deformation of the membrane, formation of the water chain, and translocation of the ions.25 Other
41 factors such as the orientation of the target molecule may also be important for complex solutes.21 In
42 order to prevent hidden energy barriers, the RC must be able to describe all these different
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44 configuration changes.
45 In this work, we design a single RC that can be used to perform umbrella sampling for the water-chain
46 assist translocation process of ions and polar groups. We show that the new RC can be successfully
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48 used to obtain continuous sample data roughly along the minimum free energy path (MFEP).
49 Furthermore, with the sample data, we can accurately estimate the one-dimensional PMF profiles based
50 on a bin-segmentation along the transition path in the phase plane of the two component of the reaction
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coordinate. This paper is organized as follows:
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53 First, we perform umbrella sampling for the permeation process of a sodium ion through a
54 dimyristoylphosphatidylcholine (DMPC) lipid membrane with a reaction coordinate 𝜉𝑝 designed in
55 the previous work.25 We show that there exists a hidden energy barrier for the formation and break of
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57 the water chain. In order to capture the hidden energy barrier, another reaction coordinate 𝜉𝑤 is
58 designed to describe the continuity of the water chain. Further analysis indicates that there is a big gap
59 on the phase plane of 𝜉𝑤 and 𝜉𝑝 between the pathway obtained by umbrella sampling along 𝜉𝑝 and
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3 the MFEP roughly obtained by relaxation dynamics from the transition state.27
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5 Next, in order to overcome the drawbacks of sample data obtained with 𝜉𝑝, we design a new RC by a
6 linear combination of 𝜉𝑝 and 𝜉𝑤. The new RC is able to describe the three types of configuration
7 changes in water-chain assist permeation process as mentioned above. Based on the new RC, we
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9 perform umbrella sampling and record the values of the RC and its two components during the
10 sampling process. With the phase plane study, we show that the trajectories obtained in umbrella
11 sampling continuously follow the MFEP, which indicates the validity of the new RC.
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Then, the PMFs as functions of the new RC are calculated using both the traditional method--weighted
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14 histogram analysis method (Wham)28 and a newly developed method--weighted least square analysis
15 method (Welsam)29 for the transmembrane permeation of the sodium ion. The PMF profiles obtained
16 using the two methods are consistent with each other. However, although our relaxation simulations
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18 from the transition state suggest an observable free energy barrier, no energy barrier for the nucleation
19 of the water chain is observed in the both profiles. In order to improve the free energy analysis result, a
20 bin segmentation of the same data is performed along the MFEP in the two dimensional phase space of
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22 the two components of the RC. The PMF is recalculated using Welsam. In this new PMF profile, a
23 clear barrier of about 0.7 kcal/mol is observed for the break of the water chain. Also, the free energy
24 barrier is about 2.4 kcal/mol lower than that in the above PMF.
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Finally, we apply the same RC and the PMF calculation method in the free energy calculations of the
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27 permeation processes of a sodium ion through membranes with different thicknesses, namely, the
28 palmitoyloleoylphosphatidylcholine (POPC) and diarachidoylphosphatidylcholine (DGPC) lipid
29 membranes, and discuss the change of energy barriers as the membrane thickness increases.
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32 Method
33 System Setup and Simulation Details
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35 To eliminate the finite-size effect of simulation systems as discussed in the previous work,25 three
36 systems of solvated membranes of 128 PC lipids with different tail lengths were built up for simulation.
37 The detailed setup of the three systems is listed in Table 1. All MD simulations were performed using
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GROMACS version 4.6.3 (www.gromacs.org)30 with NPT ensemble. CHARMM 36 with cmap
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40 all-atom lipid parameters31 and TIP3P water model32 were used in all simulations. The van der Waals
41 interactions were computed with a cut-off of 1 nm. Electrostatic interactions were calculated using the
42 particle-mesh-Ewald algorithm. Bonds involving hydrogen atoms were restrained using LINCS.33
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44 The integration time-step is 2fs. A heat bath with T=310K was applied with a time constant τT= 0.5 ps
45 using velocity-rescaling temperature coupling algorithm.34 Atmospheric pressure of 1 bar was
46 maintained using Parrinello-Rahman semi-isotropic pressure coupling method35 with the isotropic
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48 compressibility of 4.6×10-5 per bar and time constant τP= 5 ps.
49 Table 1. List of three simulations performed
50 Lipid type Number of lipid Number of pairs of Na+ Number of water
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molecules and Cl- ions molecules
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53 DMPC (14:0 PC) 128 10 4364
54 POPC (16:0-18:1 PC) 128 12 4349
55 DGPC (20:0 PC) 128 10 4480
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58 Definition of Reaction Coordinates
59 For water-chain assist paths, both leaflets of the membrane deform close to each other when the ion
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3 moves into the hydrophobic core as described in the previous works.25,36 Based on this observation, a
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5 reaction coordinate 𝜉𝑝 which represents the distance difference between the ion and the two local
6 hydrophilic-hydrophobic interfaces of the membrane is defined as
7 𝜉𝑝 = (𝑍𝑢 + 𝑍𝑏) ―2𝑍𝑖𝑜𝑛,
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where 𝑍𝑢 and 𝑍𝑏 represent the heights of the mass center of the two local interfaces of the
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10 ∑𝑖𝑍𝑖,𝑢

11 membrane. The heights are defined as 𝑍𝑢 = 𝑁𝑢

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and
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∑𝑖𝑍𝑖,𝑏
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15 𝑍𝑏 = ,
𝑁𝑏
16 where the oxygen atoms in the cylindrical local region with the radius of 1 nm are used for calculation,
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18 𝑖 is the index of the oxygen atom, 𝑢 and 𝑏 denote the upper and bottom interfaces, respectively. 𝑍𝑖
19 is the z-coordinate of the i-th oxygen atom, 𝑁 is the number of the selected oxygen atoms. 𝑍𝑖𝑜𝑛
20 represents the z-coordinate of the ion Na+. The geometrical meaning of 𝜉𝑝 is illustrated in Figure 1.
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Figure 1. Illustration of the reaction coordinate 𝜉𝑝. 𝑍𝑢 and 𝑍𝑏 represent the heights of the weighted
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40 center of mass of the two local hydrophobic−hydrophilic interfaces of the membrane. The local region
41 confined in the cylinder is represented by the dashed lines. The blue ball represents the objective ion
42 Na+ and 𝑍𝑖𝑜𝑛 represents its z-coordinate. The red balls and white balls represent the oxygen and
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44 hydrogen atoms, respectively.
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46 In addition to 𝜉𝑝, a reaction coordinate 𝜉𝑤 that describes the continuity of the water chain is designed
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by modifying the reaction coordinate in the previous work.25 In this work, 𝜉𝑤 is defined as
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49 𝜉𝑤 = 𝐿𝑤𝑏 ― 𝐿𝑤𝑢,
50 where 𝐿𝑤𝑏 and 𝐿𝑤𝑢 effectively describe the gap lengths between neighboring water molecules in the
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water chain between the ion and the bottom and the upper lipid leaflets, respectively. The oxygen
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53 atoms of lipids and water molecules inside the cylinder centered at the ion with the radius 1 nm are
54 used to calculate the gap length. We sort the oxygen atoms according to their z-coordinates and
55 calculate the differences of the z-coordinates between neighboring oxygen atoms. The gap lengths are
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57 defined as
58 16
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𝐿𝑤𝑏 = [ ∑(𝑍
𝑖,𝑏
𝑖,𝑂 ― 𝑍𝑖 + 1,𝑂)6]

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3 for the bottom part of the water chain and
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6 𝐿𝑤𝑢 = [ ∑(𝑍 𝑖,𝑂 ― 𝑍𝑖 + 1,𝑂)6]
7 𝑖,𝑢
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9 for the upper part of the water chain, where 𝑍𝑖,𝑂 is the z-coordinate of the 𝑖 ― th oxygen atom. Because
10 the power is big, the largest term of |𝑍𝑖,𝑂 ― 𝑍𝑖 + 1,𝑂| contributes the most to 𝐿𝑤𝑏 and 𝐿𝑤𝑢. Thus, 𝐿𝑤𝑏
11 and 𝐿𝑤𝑢 can be used to estimate the biggest gap length for the bottom and upper water chain,
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respectively. When the ion is in the bulk solution, 𝐿𝑤𝑏 (or 𝐿𝑤𝑢) is nearly the thickness of the
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14 hydrophobic core in equilibrium whereas 𝐿𝑤𝑢 (or 𝐿𝑤𝑏) is nearly zero, so |𝜉𝑤| is approximately a
15 constant. For a “continuous” water chain, both 𝐿𝑤𝑢 and 𝐿𝑤𝑏 are less than 0.3nm, thus |𝜉𝑤| maintains
16 a small value.
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18 The new RC is a linear composition of the two parameters
19 𝜉 = α𝜉𝑝 + (1 ― 𝛼)𝜉𝑤,
20 The parameter 𝛼 is set to be 0.4 in this study. The transition state is reached when 𝜉 is equal to zero.
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23 Relaxation process from the transition state
24 The MFEP can be roughly obtained by the relaxation dynamics released from the transition state.
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Twenty unbiased MD simulations were performed starting from the transition states. Initial
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27 configurations were randomly selected from the “transition states” obtained from a biased MD
28 simulation in which 𝜉 is controlled near to zero. The ion moved to one of the two interfaces at its
29 equilibrium states. The trajectories of the twenty relaxation processes are plotted on the phase plane
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31 with the x-axis being 𝜉𝑤 and the y-axis being 𝜉𝑝. Based on the data from the relaxation trajectories, a
32 relatively smooth curve which represents the direction of relaxation path is fitted, as shown in Figure
33 3b by the red line. The transition path is zoomed by the corresponding scale on the phase plane with the
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35 x-axis being (1 ― 𝛼)𝜉𝑤 and the y-axis being α𝜉𝑝.
36 The following steps are used to obtain a smooth curve that represents the transition path: (1) First we
37 trace out a series of centers, 𝜉0𝑖, 𝑖 = 1,2, …,𝑚, symmetrically from the relaxation trajectories on the
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phase plane by hand. (2) Then we linearly interpolate nearby centers and calculate the arc length to
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40 generate a series of approximately equally spaced points 𝜉1𝑖, 𝑖 = 1,2, …,𝑀, where 𝑀 is significantly
41 𝑖―1
42 greater then 𝑚. (3) Next we calculate 𝜉2𝑖 by the decomposition 𝜉1𝑖 = 𝜉2𝑖 + 𝑀 ― 1(𝜉1𝑀 ―𝜉11). Denote
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44 the Fourier transform of 𝜉2𝑖 by 𝜉2(𝜔), 𝜔 = 0, 1, …, 𝑀 ― 1, then we calculate 𝜉3𝑖 by the inverse
45 Fourier transform of 𝜉2(𝜔)𝑒𝐷𝜔(𝜔 ― 𝑀), where 𝐷 is a smooth factor. (4) Finally, we obtain a smooth
46 𝑖―1
47 curve represented by 𝜉𝑖 = 𝜉3𝑖 + 𝑀 ― 1(𝜉1𝑀 ―𝜉11). In the current case, we have made use the fact that the
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49 transition path in this work has a same slope at its two ends due to the symmetry. When the slopes at
50 the two ends are different, there is a boundary effect in step (4). In this case, we may use linear
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extrapolation to obtain more points in 𝜉1𝑖, so that the points we really use are far away from the
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53 boundary.
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55 Umbrella sampling
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57 1
In our umbrella sampling, bias potentials 𝑈𝑖（𝜉） = 2𝐾𝑖(𝜉 ― 𝜉𝑖)2 were used to confine the system
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59 around the pre-given target values of reaction coordinates, 𝜉𝑖, where 𝑖 is the index of the 𝑖 ― th
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3 sampling window and 𝐾𝑖 is the elastic modulus. The force component 𝑓𝑗 induced by the harmonic
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5 ∂𝜉
potential can be calculated as 𝑓𝑗 = ― 𝐾𝑖(𝜉 ― 𝜉𝑖)∂𝑥𝑗. To implement the forces, the pull code has been
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7 modified in standard GROMACS 4.6.3, that is available from the authors upon request.
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Umbrella sampling was performed using 𝜉𝑝 as the reaction coordinate in 30 windows over the
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10 sampling range [−5.2, 0] for the DMPC system. To ensure that the transition state is well sampled, a
11 relatively stiff elastic modulus 𝐾𝑖 = 600 kJ·mol-1·nm-2 and a window width of 0.1 nm were used in the
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transition state region [−0.5, 0] while a relatively soft elastic modulus 𝐾𝑖 = 300 kJ·mol-1·nm-2 and a
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14 window width of 0.2 nm were used in the outer region [-5.2,-0.6]. In each window, it was equilibrated
15 for 10 ns and then simulated for 50 ns.
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Umbrella sampling was performed using 𝜉 as the reaction coordinate in 47 windows over the
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18 sampling range [−4.0, 4.0] for the DMPC system, 49 windows over the sampling range [−4.4, 4.4] for
19 the POPC system and 53 windows over the sampling range [−4.8, 4.8] for the DGPC system. To ensure
20 that the transition state is well sampled, a relatively stiff elastic modulus 𝐾𝑖 = 600 kJ·mol-1·nm-2 and a
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22 window width of 0.1 nm were used in the transition state region [−0.5, 0.5] for DMPC, [-0.4,-0.4] for
23 DGPC, [-0.3, 0.3] for POPC while a relatively soft elastic modulus 𝐾𝑖 = 300 kJ·mol-1·nm-2 and a
24 window width of 0.2 nm were used in the outer region. In each window, it was equilibrated for 20 ns
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and then simulated for 60 ns.
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28 PMF calculation along the new RC using Wham and Welsam
29 The PMFs were constructed from the sample data with both Wham28 and Welsam29. The sampling
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31 ranges of each system are divided into histogram bins with equal length, namely, 400 bins for the
32 DMPC system, 457 bins for the POPC system, and 495 bins for the DGPC system. Wham was
33 performed with a tolerance of 1x10-5.
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36 PMF calculation along the transition path using Welsam
37 The data of both α𝜉𝑝 and (1 ― 𝛼)𝜉𝑤 were recorded during umbrella sampling. As is shown in Figure
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4a, the sampling points were mapped onto the two dimensional phase plane. The trajectories from the
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40 umbrella sampling highly coincide with the relaxation transition path.
41 The data from umbrella sampling were distributed into bins with equal length along the transition paths
42 for all three systems. The number of bins is 400 for DMPC, 457 for POPC, and 495 for DGPC. The
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44 data far away from the transition path were excluded in the free energy analysis. For example, the
45 sample points of non-water-chain configurations (when (1 ― 𝛼)|𝜉𝑤| is more than 0.2nm) were
46 excluded from the bins in the transition state region to calculate the free energy of water-chain
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48 configurations. The free energy of each bin was calculated with Welsam29. The errors were estimated
49 by the differences of free energies above and below the membrane center due to the symmetry of the
50 permeation process, 𝑒𝑟𝑟𝑜𝑟 = |𝑃𝑀𝐹(RC) ― 𝑃𝑀𝐹( ― RC))| 2.
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53 Results
54 Umbrella sampling along 𝝃𝒑 leads to sampling errors
55 It has been shown that there are severe hysteresis and hidden energy barriers in the umbrella sampling
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57 of the permeation process of a charged group through a lipid membrane using the z-distance as the
58 reaction coordinate.25 An improved reaction coordinate 𝜉𝑝 has been designed in the previous work to
59 describe the permeation process of charged methyl guanidine.25 Based on the umbrella sampling with
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3 𝜉𝑝, a water-chain assist mechanism has been discovered.
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5 The permeation trajectories of a sodium ion through DMPC membrane are obtained from the umbrella
6 sampling along the reaction coordinate 𝜉𝑝. As 𝜉𝑝 increases from -5.2nm to -0.8 nm, the ion moves
7 from the bulk solution to the hydrophobic core of the membrane. When the ion approaches the center
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9 of the membrane, the monolayer connecting to the ion undergoes a large deformation to prevent the
10 sodium ion from exposing to the hydrophobic core. In the windows when 𝜉𝑝 is about -0.6 nm,
11 formation and break of a water chain are observed as shown in Figure 2. However, only five times of
12
configuration jumps were observed during the 50 ns simulation in one window, which indicates a
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14 considerable free energy barrier. Note that using 𝜉𝑝 is not able to distinguish the two configurations
15 with and without a water chain. Hence, a new reaction coordinate 𝜉𝑤 is introduced to describe the
16 continuity or break of the water chain.
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18 In Figure 3a, the umbrella sampling data obtained with the reaction coordinate 𝜉𝑝 are projected onto
19 the phase plane of the two reaction coordinates, 𝜉𝑤 and 𝜉𝑝. As can be seen from the detailed view in
20 Figure 3a, there is a region with sparse sample data where 𝜉𝑤 is about -0.3nm, which suggests a free
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energy barrier in the direction of 𝜉𝑤. This kind of hidden energy barrier can lead to errors in free
22
23 energy analysis because the states on the two sides of this energy barrier can have different free
24 energies but have the same reaction coordinate 𝜉𝑝.
25
The transition state where the ion is located in the middle of the membrane core can be obtained in the
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27 umbrella sampling by confining 𝜉𝑝 to be near zero. We performed the unbiased relaxation dynamics
28 in which the initial configuration is randomly selected from the transition states. The trajectories of
29 relaxation processes starting from the transition state are also mapped on the same phase plane by the
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31 blue points (Figure 3b). In general, the trajectories from the transition state to the equilibrium state
32 roughly follow the MFEP. A symmetric relaxation trajectory is obtained due to the symmetry of the
33 membrane. The representing transition path is traced out by smoothly fitting the relaxation trajectories
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35 on the phase plane as shown in Figure 3b by the red line. By comparing the path of umbrella sampling
36 (traced out as the white line in Figure 3a) with the MFEP, we observe a big gap between the two paths.
37 In particular, in the umbrella sampling with 𝜉𝑝, the water chain begins to form only when |𝜉𝑝| is as
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small as 1 nm, while along the MFEP, the formation and break of the water chain occur when |𝜉𝑝| is
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40 about 1.7 nm.
41 In order to overcome the two drawbacks, namely the discontinuity of the sample data and the gap
42 between the sample path and MFEP, we proceed to design a new reaction coordinate.
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54 Figure 2. Two configurations are observed with and without a water chain across the membrane in the
55 window when 𝜉𝑝 is confined to be about -0.6 nm.
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23 Figure 3. (a) Sample data from the simulation using 𝜉𝑝 as the reaction coordinate projected on the
24 phase plane of 𝜉𝑤 and 𝜉𝑝. A region with sparse sample data when 𝜉𝑤 is about -0.3nm is observed. In
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addition, the sample path (white line) deviates from the relaxation transition path (red line) near the
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27 transition state. (b) Relaxation trajectories from the transition state. The detailed view in Figure 3b
28 shows the region with sparse data corresponding to the nucleation process of water chains.
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32 Umbrella sampling along the new reaction coordinate
33 In order to reduce the sampling errors and obtain an accurate PMF, a new reaction coordinate 𝜉 is
34
35 designed as 𝜉 = α𝜉𝑝 + (1 ― 𝛼)𝜉𝑤, which is appropriate for umbrella sampling of the whole process of
36 water-chain assist permeation across lipid membranes of ions.
37 In Figure 4a, the data obtained from umbrella sampling with 𝜉 are projected onto the phase plane of
38
the two components of the new reaction coordinate, (1 ― 𝛼)𝜉𝑤 and α𝜉𝑝. A symmetric permeation
39
40 path is obtained from our umbrella sampling. In particular, a water bridge forms smoothly as 𝜉
41 approaches zero controlled by the umbrella potentials. The permeation process can be roughly divided
42 into five symmetric stages (from A to E). In stages A and E, the sodium ion Na+ moves towards (or
43
44 away from) the head group layer of the lipid membrane from (towards) the bulk solution. During these
45 stages, the value of 𝜉𝑤 does not change significantly. In stages B and D, both leaflets of the membrane
46 deform towards the membrane core to maintain a smaller value of |𝜉| while keeping the ion solvated
47
by water molecules and lipid head groups. During these stages, the value of 𝜉𝑝 changes linearly with
48
49 the value of 𝜉𝑤. As |𝜉| approaches zero, a water chain is formed across the membrane. In stage C, a
50 full water chain is maintained as suggested by the small value of 𝜉𝑤. In particular, the transition state is
51
obtained when 𝜉 is equal to zero. At the transition state, the ion is located at the center of the
52
53 membrane on the water chain. From Figure 4a, we can see that the transition path traced out from
54 relaxation dynamics can also represent well the data obtained in umbrella sampling. This further
55 verifies the validity of the new RC.
56
57 We note that the curve is almost horizontal at the configuration transition state from stage B to C (or
58 stage C to D), which corresponds to the formation (or break) of the water chain. Similar permeation
59 processes have been observed for other cationic solutes in previous works.13,25,36 As is shown by the
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3 detail view in Figure 3b, the relaxation trajectories are very sparse near these transition states, which
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5 suggests a considerable free energy barrier. This energy barrier has also been discussed in previous
6 studies.23–25 Similar results for System POPC and DGPC are shown in Figure S3.
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40 Figure 4. Results of umbrella sampling with 𝜉 for system DMPC. (a) Sample data in the phase plane
41 of the two components of the 𝜉. The phase plane is devided into five domains along the transition path.
42 The detail view shows that there are sufficient sample data at the transition state for water chain
43
44 nucleation, where the transition path is approximately horizontal. (b) Typical MD snapshots obtained
45 in umbrella sampling from the equilibrium state to the transition state. The sodium ion Na+ is shown in
46 blue. The lipid oxygen atoms are shown by red spheres and the phosphorus atoms as tan spheres. The
47
48 water molecules are shown by small red and white sticks. The notations A/B and B/C denote the states
49 at the boundaries between the corresponding domains.
50
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Free energy calculation based on the new RC
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53 The PMF profile obtained with umbrella sampling using Wham28 and Welsam29 along the new RC is
54 shown in Figure 5a. In these free energy calculations, the bin segmentation is simply based on the
55
recorded value of 𝜉 of the sample data. The two curves match each other very well. The free energy
56
57 barrier (relative free energy difference between the transition state and the equilibrium state in the bulk
58 solution) is about 19.0 kcal/mol. Two local minimums with a depth of about 0.9 kcal/mol are observed
59 at |𝜉|≈2.8 nm, demonstrating the adsorption of cations to lipid membranes. The PMF profile is
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3 approximately symmetrical and the statistical errors are less than 0.9 kcal/mol. Similar results are
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5 shown in Figure S4 for system POPC and DGPC.
6 However, no free energy barrier for the formation (break) of a water chain is observed in the PMF
7 profile in Figure 5a, which is inconsistent with the above discussion. This disparity can be attributed to
8
9 the fact that the bin segmentation based on the value of the 𝜉 cannot represent the transition direction
10 at the transition state for the formation (break) of water chains.
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28 Figure 5. PMF profiles. (a) PMF profiles obtained with the simple bin segmentation based on the value
29
of 𝜉. The blue and red curves are obtained with the free energy analysis method Wham and Welsam,
30
31 respectively. The free energy barrier for the permeation process is 19.0 kcal/mol and the depth of the
32 two minimums corresponding to the membrane adsorbed states is 0.9 kcal/mol. (b) The PMF profile
33 computed along the transition path using Welsam. A clear barrier of about 0.7 kcal/mol is observed for
34
35 the break of the water chain. The free energy barrier is 16.6kcal/mol, and the depth of the two
36 minimums is 2.3 kcal/mol. A, A/B, B, B/C and C denote the domains and domain boundaries shown in
37 Figure 4a.
38
39
40 Free energy calculation along the transition path
41 In order to overcome the drawbacks of the above bin segmentation, we perform a bin segmentation
42 along the transition path fitted by the relaxation trajectories in the phase plane of α𝜉𝑝 and (1 ― 𝛼)𝜉𝑤,
43
44 as shown in Figure 6.
45 Since the umbrella potential can no longer be regarded as a constant in each bin in this case, it is not
46 convenient to calculate the PMF profile using the traditional free energy analysis method Wham. In the
47
48 newly developed free energy analysis method Welsam29, the umbrella potential need not to maintain a
49 close value in each bin. In other words, the bin segmentation is decoupled from the application of
50 umbrella potential. The PMF profile along the transition path is calculated using Welsam and shown in
51
Figure 5b.
52
53 In the new PMF profile, the free energy barrier becomes 16.6 kcal/mol, which is 2.4 kcal/mol lower
54 than that in Figure 5a. The depth of the free energy well for the membrane adsorbed state becomes 2.3
55 kcal/mol, which is about 1.4 kcal/mol deeper than that in Figure 5a. Remarkably, a clear barrier of
56
57 about 0.7 kcal/mol is observed in the break of the water chains region. With the new bin segmentation,
58 the symmetry of the PMF profile is improved and the statistical errors become less than 0.5 kcal/mol.
59 Similar results for system POPC and DGPC are shown in Figure S5.
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Figure 6. Bin segmentation along the transition path. 400 bins are equally partitioned along the
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27 transition path. The lines show the boundary of neighboring bins which are perpendicular to the
28 transition path.
29
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31 Figure 7 can also be helpful to understand the big gap between the transition paths traced out from the
32 umbrella sampling data along 𝜉𝑝 and the relaxation trajectories depicted in Figure 3. The free energy
33 barrier for the formation of a water chain is nearly 5.0 kcal/mol, whereas the barrier for the break of
34
that is less than 1.0 kcal/mol when 𝜉𝑝 is controlled to be near -1.7nm. As a result, only the
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36 non-water-chain state is sampled by controlling 𝜉𝑝 in the nearby windows, which leads to the big gap
37 of the two paths.
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55 Figure 7. The free energy values of the water-chain configuration, non-water-chain configuration and
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57 the transition state (red star) are labelled along the transition path. The free energy barrier of formation
58 a water chain is nearly 5.0kcal/mol, while the barrier for the break of the water-chain is less than
59 1.0kcal/mol.
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5 Effects of membrane thickness
6 To further investigate the effect of membrane thickness, we calculate the free energy curves of the
7 permeation processes through two thicker membranes, namely, POPC and DGPC membranes. The
8
9 hydrophobic thickness is defined by the bilayer's hydrocarbon acyl chains. In agreement with previous
10 experimental measurement, the average values of DMPC, POPC, DGPC bilayers are 2.56, 2.87 and
11 3.23 nm from our simulations.37 As shown in Figure 8 and Table 2, all the three free energy curves
12
have very similar characteristics. They all contain two small free energy barriers (less than 1.1kcal/mol
13
14 for all three systems) in the break of water chain regions and two local minimums for membrane
15 adsorbed state of cations. The free energy barrier for the whole permeation process increases with the
16 hydrophobic thickness. Nevertheless, this increase is not linear: although the difference in thickness
17
18 between DMPC and POPC membranes is almost the same as that between POPC and DGPC
19 membrane, the difference of energy barriers differs a lot.
20 It is of interest to see that once the water chain is formed, the free energy costs to move the ion to the
21
22 center are similar for the three systems as suggested by Figure 8 and Table 2. This is mainly due to the
23 fact that the average lengths of stable water chains are similar for the three systems as observed in our
24 umbrella sampling. On average, the water chains are thinner in thicker membranes, thus easier to be
25
broken under thermal fluctuations. In Table 2, we statistically estimated two time scales from the
26
27 relaxation processes discussed above. 𝑡𝑟 is the relaxation time, which is defined as the duration that
28 the ion moves from the middle of the water chain to the hydrophobic-hydrophilic interfaces until its
29 z-coordinate is more than 𝑍𝑢 or less than 𝑍𝑏. 𝑡𝑤 is the time for the water chain to be broken, which is
30
31 defined as the timescale of the relaxation process from the intact water chain at the transition state to a
32 broken one with the values that 𝐿𝑤𝑢 or 𝐿𝑤𝑏 is more than 0.4 nm. The data indicates that the water
33 chain in DMPC membrane is more stable than in other membranes. By comparing the values of 𝑡𝑟
34
and 𝑡𝑤, we can find that the water chains are not broken until the ion reaches the interface in system
35
36 DMPC, while the water chains have been broken before the ion moves to the interfaces in System
37 POPC and DGPC.
38
The permeability, P, of ions through the three lipid membranes is estimated by
39
ℎ
40 P≈ Δ𝐺 ,
41 𝑡𝑟𝑒
𝑘𝐵𝑇

42 where ℎ is the hydrophobic thickness, 𝑡𝑟 is the relaxation time, Δ𝐺 is the free energy barrier, 𝑘𝐵 is
43
44 the Boltzmann constant and T=310K is the temperature. The permeability coefficient of system DMPC
45 is on the same order as the experiment data of permeability of potassium ion,8 but appears to be smaller
46 than the experimental data for system POPC and DGPC.5,8
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19 Figure 8. The PMF profiles of the three systems align along the minimum. The PMF exhibits a clear
20 pore nucleation barrier of 0.6~ 1.1 kcal/mol at the water chain formation/break region.
21
22
23 Table 2. Results of the PMF barriers (Δ𝐺), the barriers for the break of water chain (Δ𝐺𝑤), the barriers
24 of translocation of the ion to the center after the water chain is formed (Δ𝐺𝑐), the membrane
25
hydrophobic thickness (ℎ), the relaxation time of the ion from the transition state to interface (𝑡𝑟), the
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27 life time of water-chain during the relaxation processes (𝑡𝑤), and the permeability coefficient (P) for
28 the three systems.
29 𝚫𝑮𝒘 𝚫𝑮𝒄 𝒕𝒓 (ns) 𝒕𝒘 (ns)
𝚫𝑮 (kcal/mol) (kcal/mol) (kcal/mol) 𝒉 (nm) 𝐏 (cm/s)
30
31 DMPC 16.6 0.7 6.2 2.56 8.27 10.93 6.55*10-10
32 POPC 24.4 0.6 6.3 2.87 3.86 1.51 4.28*10-15
33 DGPC 29.3 1.1 6.7 3.23 2.64 0.95 2.46*10-18
34
35
36 Discussion
37 Suitable reaction coordinates are crucial for free energy calculation. In this paper, we have designed a
38
39 single RC to describe the water-chain assist permeation process of ions through lipid membranes. The
40 new RC is a linear composition of two components, where the first component is used to describe the
41 deformation of the lipid membrane and the translocation of the ion whereas the second component is
42
used to describe the continuity of the water chain. Since the new RC can be used to describe the three
43
44 types of configuration changes, it has the potential of describing general water-chain assist
45 transmembrane translocation processes. As an evidence, the same RC is also successfully used in
46 systems with different lipid bilayers. When other configuration changes, such as the orientation
47
48 changes of solutes, are also important in the translocation processes, additional RCs may be combined
49 with the new RC designed in this work to perform free energy calculation.
50 Umbrella sampling along the new RC is successfully used to obtain continuous sample data roughly
51
52 along the MFEP. However, as our phase diagram analysis shows, the direction of the MFEP deviates
53 from the direction of the RC at a few states. These deviations introduce errors in the PMF profile based
54 on the bin segmentation simply according to the value of the RC. In order to obtain an accurate PMF
55
profile, we perform a careful bin segmentation in the two dimensional phase space along the transition
56
57 path. In this new PMF profile, details are captured accurately including the energy barrier for the break
58 of the water chain and the membrane adsorption energy. The free energy barrier for the permeation
59 process also significantly decreases compared to that obtained with the simple bin segmentation. From
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3 this point of view, it is important to record the two components of the RC in umbrella sampling in
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5 order for a proper bin segmentation.
6 Two free energy analysis methods are applied in this work. For the simple bin segmentation, both
7 Wham and Welsam can be used to calculate the PMF profiles. The two curves match each other very
8
9 well. For the improved bin segmentation, Wham cannot be used directly since the umbrella potential
10 can differ a lot in a single bin. The newly developed free energy analysis method Welsam shows its
11 potential in obtaining one-dimensional PMF profiles for complex systems due to its flexibility.
12
Therefore, under the framework of Welsam, a good RC is only required to apply umbrella potentials
13
14 with to obtain continuous sample data along the transition path. The accurate one-dimensional PMF
15 profiles can be calculated along the transition path with the careful bin-segmentation along the
16 transition path in a relatively high dimensional phase space.
17
18 As shown by our study, the free energy barrier for the permeation process increases with membrane
19 thickness. In previous papers38–40, dehydration path is proposed for the permeation process through
20 thick membranes. The free energy barrier for the dehydration path is believed to be nearly independent
21
22 of membrane thickness. This suggests that for a membrane with a very large thickness, the dehydration
23 mechanism will have a lower free energy barrier. However, in our umbrella sampling simulations,
24 when the RC is confined to be close to zero, the dehydration configuration is not observed. Thus we
25
believe that the water-chain assist mechanism is the dominant mechanism for transmembrane
26
27 permeation of cations through DMPC, POPC, and DGPC membranes.
28
29
Conclusion
30
31 In this work, we have designed an effective reaction coordinate for free energy calculations of water
32 chain assist transmembrane permeation processes of ions and highly polar groups. The new reaction
33 coordinate is a linear combination of two components. Continuous sample data along the minimum free
34
35 energy path are obtained by umbrella sampling along the new reaction coordinate. Although the new
36 reaction coordinate is helpful in performing one-dimensional umbrella sampling, it is still not perfect in
37 describing the formation and break of membrane-spanning water chains in free energy analysis.
38
Therefore, a careful bin segmentation along the transition path in the phase plane of the two
39
40 components of the new reaction coordinate is performed to obtain accurate one-dimensional PMF
41 profiles. The one-dimensional PMF profiles can accurately reflect the free energy changes of
42 membrane adsorption of ions and nucleation of a water chain. Therefore, the new reaction coordinate
43
44 can be successfully used to perform a one-dimensional free energy calculation, although a bin
45 segmentation in the two-dimensional phase plane of the two components is still necessary. With this
46 one-dimensional free energy calculation, the complexity and computational cost can be reduced greatly
47
48 in real applications.
49
50 Acknowledgement
51
Dan Hu was supported by NSFC grants 91630208 and 11471213, the NYU Abu Dhabi Institute under
52
53 grant G1301, and HPC π at Shanghai Jiao Tong University. Dong-Qing Wei was supported by the
54 grants from the Key Research Area Grant 2016YFA0501703 of the Ministry of Science and
55 Technology of China, the National Natural Science Foundation of China (Contract no. 61832019,
56
57 61503244), the State Key Lab on Microbial Metabolism, and Joint Research Funds for Medical and
58 Engineering & Scientific Research at Shanghai Jiao Tong University (YG2017ZD14).
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5 Supporting Information
6 The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.
7
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22 (39) MacCallum, J. L.; Bennett, W. F. D.; Tieleman, D. P. Transfer of Arginine into Lipid Bilayers
23 Is Nonadditive. Biophys. J. 2011, 101 (1), 110–117.
24 (40) Li, L.; Vorobyov, I.; Allen, T. W. The Different Interactions of Lysine and Arginine Side
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Chains with Lipid Membranes. J. Phys. Chem. B 2013, 117 (40), 11906–11920.
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23 The single reaction coordinate can be used to perform one-dimensional umbrella sampling for
24 transmembrane permeation of ions. The sample data are continuous along the transition path. By
segmentation of bins along the transition path, an accurate curve for the potential of mean force is obtained
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26 (break) of transmembrane water bridges are well captured by this free energy analysis.
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