Nano-confinement of biomolecules: Hydrophilic …274 Nano Res. 2016, 9(2): 273–281 by walls of a...

Nano-confinement of biomolecules: Hydrophilicconfinement promotes structural order and enhancesmobility of water molecules

Bachir Aoun1 and Daniela Russo2,3 ()

1 Argonne National Laboratory, Chicago, IL 60439, USA 2 CNR-IOM, c/o Institut Laue-Langevin, 38400 Grenoble, France 3 Institut Lumière Matière, Université de Lyon, 69622 Lyon, France

Received: 13 May 2015 Revised: 28 September 2015 Accepted: 4 October 2015 © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2015 KEYWORDS nano-confinement, protein folding, hydration water, carbon nanotube, drug delivery

ABSTRACT Molecular dynamics simulations have been used to investigate the confinementpacking characteristics of small hydrophilic (N-acetyl-glycine-methylamide, Nagma) and hydrophobic (N-acetyl-leucine-methylamide, Nalma) biomoleculesin large diameter single-wall carbon nanotubes (SWCNTs). We find that hydrophilic biomolecules easily fill the nanotube and self organize into ageometrical configuration which reminds the water structural organization under SWCNT confinement. The packing of hydrophilic biomolecules insidethe cylinder confines all water molecules in its core, which enhances theirmobility. Conversely, hydrophobic biomolecules accommodate into the nanotubeswith a trend for homogeneous filling, which generate unstable small pockets ofwater and drive toward a state of dehydration. These results shed light on keyparameters important for the encapsulation of biomolecules with direct relevancefor long-term storage and prevention of degradation.

1 Introduction

A better understanding of the behavior of confined proteins has significant value for practical applications, as it can provide direct insight into protein folding and structural stability. Proteins and enzymes under confinement, or entrapment, can have enhanced structural stability, which can be used to improve their performance and allow for adaptation to specific industrial and diagnostic applications, such as, drug

delivery, biocatalysis, and biosensors [1]. The encap-sulation of individual biomolecules can be encap-sulated, also prevent potential self-aggregation, and be used to isolate particular allosteric conformational states. This can reduce microbial degradation, improve storage longevity, and produce better drug preservation and delivery.

Confinement can be achieved by the presence of other stable macromolecules (proteins or polymers) at sufficiently high concentration. It can also be induced

Nano Research 2016, 9(2): 273–281 DOI 10.1007/s12274-015-0907-7

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274 Nano Res. 2016, 9(2): 273–281

by walls of a cage within a silica matrix, within pores or tubes, or simply by the application of external non-denaturing pressure. Recent development of new methods of solubilization [2, 3] has created new opportunities for biological application of carbon nano-tubes (CNT) allowing for their emergence as a new material to achieve nano-confinement. Single-walled carbon nanotubes (SWCNTs) are hollow, cylindrical structures created by interlinked networks of carbon atoms, with nanoscale dimensions that provide large inner volumes capable of high cargo loading within the cylindrical tube.

The application of CNT has been explored in numerous biochemical studies. CNT are particularly attractive for their ability to spontaneously encapsulate or adsorb on their sidewalls, proteins, peptides, DNA fragments, and other biomolecules. Functionalization of CNT can allow them allow to cross the cell membranes and to accumulate in the cytoplasm low risk of toxicity, thus making them applicable to use in molecular transport and protein delivery [4, 5].

Previous work using molecular dynamics (MD) simulations to investigate the effects of geometry on nano-sized confinement of biomolecules suggest a correlation between confinement geometry and the properties of protein-folding transition states that can enhance folding rates and improve stability [6]. The role of water in the stabilizing confined biomolecules has also been studied by MD simulations. Sorin and Pande [7] observed that the addition of water solvation produced helix stability of a polypeptide chain that decreased as a function of SWCNT tube diameter, suggesting that the solvent properties play a signifi-cant role in the thermodynamics of secondary structure formation in confined spaces. Other investigations have examined the static and dynamical properties of different helix-and beta-hairpin sequences formed under CNT confinement, and reveal the subtle inter-play between primary sequences, nanotube-peptide interactions and SWCNT diameter [8, 9]. A study looking at retinal Retinal chromophore confinement in SWCNT allowed a unique insight into the role of cis/tran isomerization that triggers the biological activity of photoreceptor cells [10]. The static and dynamic properties of water confined within SWCNTs have also been successfully investigated by X-ray and neutron

scattering experiments which reveal anomalous soft dynamics, the presence of layered structures and chainlike configurations [11, 12]. The unique charac-teristics and mechanical properties of CNT make them excellent environments for investigating the effects of confinement on molecules of biological relevance, and also allow for investigation of the subtle effects of water solvation.

In the present work we address the question of how SWCNT confinement may be affected by the hydrophilic or hydrophobic biomolecules. We focus our examination on the role of specific interactions and control for effects related to molecular size. In addition, we seek to understand the local behaviors of hydration water, which are highly relevant to many biochemical processes. To better understand the specificity of biomolecule properties and water mole-cules under nano-confinement, we used a simplified protein-model biomolecules, with distinct hydrophilic and hydrophobic characteristics. Within this general framework, we first performed molecular dynamic simulations in order to investigate confinement packing of small hydrophilic and hydrophobic biomolecules in aqueous solutions. We examine: a) N-acetyl-leucine- methylamide (Nalma), which comprises a hydrophobic amino acid side chain, (CH3)2–CH–CH2, attached to the Cά atom of a polar blocked polypeptide backbone with CH3 end-caps, (CH3–CO–NH–CάH–CO–NH–CH3) and b) N-acetyl-glycine-methylamide (Nagma), which comprises the polar blocked backbone and a hydrogen atom attached to the Cά atom. These two peptides share a quite similar chemical composition with similar ensemble-averaged size; thus, they allow for a straightforward comparison between the effects of hydrophilic versus hydrophobic properties [13–20]. We investigate the structural properties of confine-ment packing in SWCNT for both biomolecules. Our findings shed light on key parameters relevant for the storage and conservation of biomolecules in both the presence and absence of water-molecule solvation.

2 Computational details

MD simulations were performed using NAMD pac-kage [21] with all-atom dihedral potential corrected variant CHARMM22/CMAP force field [22, 23], using

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275 Nano Res. 2016, 9(2): 273–281

TIP3P model. This selection of potential energy functions reflects the best combination to reproduce both structure and dynamics experimental results of Nalma and Nagma solution at equivalent con-centrations ratios [13–20]. We briefly summarize our computational approach here. A full description of the computational strategy is provided in the Electronic Supplementary Material (ESM).

We explored two amorphous randomly generated systems containing SWCNT with a 4 nm diameter and a length of 5 nm. 18,000 water molecules were used along with 600 molecules of Nalma and 900 molecules of Nagma which correspond to concentrations of 2 and 3 M, respectively. A cubic box length of 42.4 Å was used in the case of Nalma and 47.4 Å for Nagma (Table S2 in the ESM). The SWCNTs were partially filled with water and biomolecules as initial conditions (Fig. S1 in the ESM). The whole system was first equili-brated at 350 K, in the isothermal–isobaric (NPT) ensemble, for 78 ns at 1 bar. To confirm that the equili-bration procedure produces independent configurations from the initial conditions, the filling density inside the SWCNT was analyzed and will be discussed in the next section. To prepare the trajectories at 300 K, the 350 K equilibrated configurations were cooled down at a rate of 10 K/ns, followed by 25 ns of equilibration time. Finally, 5 ns of production-run dynamics were carried out at 300 K and analyzed (results described in the next section).

3 Results and discussion

We describe here Nagma and Nalma, during the structural and dynamical properties of the confined hydrophilic and hydrophobic molecules, during the final equilibration and filling stage.

Preliminary molecular dynamics simulations focused on the relation between SWCNT diameter and com-plete filling of the inner cavity.

Results indicated the existence of a minimum diameter of 1.5 nm necessary for migration of biomole-cules inside the nanotube. In order to more carefully investigate the correlation between confinement and hydrophilic/hydrophobic molecular characteristic, only MD trajectories obtained from SWCNT with a diameter of 4 nm were analyzed (see the ESM). This

relatively large diameter is compatible with high numbers of biomolecules be able to migrate inside the SWCNT cavity and thus able to achieve a complete filling. SWCNT with large diameters are also easily available for sample preparations. Density distribution functions, numbers of confined molecules, mean shell thickness and mean square displacements were calculated and are compared below.

Both peptides fill the SWCNT in the time range of 20–50 ns, and arrive at highly stabile configurations after confinement. We estimate that, after 70 ns of equilibration, 80% of the total molecules filling the SWCNT cavity are the hydrophilic Nagma while the remaining 20% are water molecules. Similar behavior is observed for the hydrophobic Nalma which fills 95% of the SWCNT internal cavity with only 5% occupied by water molecules.

Figure 1 shows the evolution of SWCNT filling as a function of time (equilibration step) for the hydro-phobic Nalma, hydrophilic Nagma, and associated water molecules. Figure 1(a) depicts the percentage of confined molecules close to the SWCNT inner walls, and shows that after 20 ns water molecules are replaced by biomolecules and that the bio-layer is stable over time. For each SWCNT atoms the calculation considers the closest neighboring atoms, it assigns the type of molecule and estimates the total percentage for the whole SWCNT. Figure 1(b) illustrates the evolution of the filling in side SWCNT indicating that a stable equilibrium is reached between 60 and 80 ns. In both cases, filling seems to take place from SWCNT walls towards the center of the tube. The space close to inner walls is filled in priority, demonstrating a preferential and stabilizing peptide-nanotube interaction. More evident in Fig. 1(b) than in Fig. 1(a) is that Nalma biomolecules fill SWCNT faster (but of the same order of magnitude) than Nagma molecules. It is worth mentioning that the average sizes of each biomolecule are similar, with Nalma containing a branch point from which extends a 4 Å long side chain. In the presence of the hydrophobic Nalma, water molecules expelled more efficiently than in presence of hydrophilic Nagma and their percentage continues to decrease slowly even at 80 ns, while it reaches a stable equilibrium around 40 ns in the second case.

Figure 2 displays number density as a function of simulation time, which is defined as the ratio of the


276 Nano Res. 2016, 9(2): 273–281

Figure 1 Evolution, as a function of time, of the carbon nanotube filling by hydrophobic Nalma (open circles), hydrophilic Nagma (open squares) and related water molecules (full circles, full squares). (a) Percentages of confined molecules close to the inner SWCNT walls. (b) Percentages of the confined molecules in the inner cavity.

numbers of atoms of a particular molecule found inside a defined space and the volume of this space (ESM for more detailed description). The behavior of the number density of all species inside the SWCNT, confirms that once the filling capacity is reached at equilibrium, the number of confined water molecules is in general smaller than the number of biomolecules and that, in the hydrophobic case, it is almost neg-ligible. The differing amounts of water contained within the SWCNT and the higher density of hydrophobic molecules can be explained by the stronger interaction between the peptide and SWCNT nanotube in the case of Nalma. Even though the hydrophobic peptides

Figure 2 Number density, calculated during the 5 ns production run, for hydrophobic Nalma (open circles), hydrophilic Nagma (open squares) and related water molecules (full circles, full squares). The calculation reveals that water density is lower than the biomolecules densities and that hydrophobic Nalma is more densely packed than hydrophilic Nagma.

exist at higher concentration in the water solution, they find more energetically favorable environments within the SWCNT, as this allows them to minimize their interaction with water. The slightly shorter filling time for Nalma can be associated with a lowering of the energy of the whole system, in agreement with the idea that molecular traffic jams at the extremities of the SWCNTs enhance the dynamics across them. This hypothesis is supported by a new concept pro-posed to explain why water molecules move faster across nano-channels [24]. Nonetheless, we are aware that this result can be related to the size of the SWCNT diameter.

To investigate the impact of the peptide encapsula-tion on structural stability and geometrical correlation properties, together with its hydration water, we calculate the cylindrical density distribution (CDD) (Fig. 3). The CDD is defined as the local density of atomic species calculated within cylindrical shells (of thickness 0.01 nm) of variable radii (from 0.01 to 2 nm) divided by the total density of the same atomic species contained inside the whole cylinder (see ESM for detailed definition). MD results show that confinement of bio molecules inside the SWCNT has a strong impact on local structures, depending on the hydrophilic or hydrophobic properties of biomolecular species. The density profile of the hydrophobic Nalma


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Figure 3 Cylindrical density distribution for hydrophobic Nalma (open circles), hydrophilic Nagma (open squares), and related water molecules (full circles, full squares). The homogeneous filling by Nalma stands in sharp contrast to the well-structured packing of Nagma. The confined water molecules also show pronounced differences in their density profiles, which are correlated to the biomolecular distributions.

clearly shows a very homogeneous filling of the SWCNT, from the center up to almost 0.3 nm from the inner wall, which however leaves an empty gap between the biomolecules and the SWCNT wall. The amount of water is negligible compared to that of the peptides and the profile suggests the presence of trapped pockets of water molecules within the packed peptide. The hypothesis for the presence of small pockets of water is supported by the shoulder observed in the water density distribution between 0.4 and 0.8 nm. As previously shown in Fig. 1(b), confined water molecules in presence of hydrophobic bio interface slowly escape from the SWCNT cavity, which suggests a slow trend toward dehydration.

Also seen in Fig. 3 is the CDD for the hydrophilic Nagma, in which the water molecules show a highly structured encapsulation and the presence of con-finement. The water appears to accommodate and fill the center of the SWCNT, with a maximum density at 0.3 nm and a progressive decrease extending to approximately 1.2 nm. There is a noticeable small peak at 1.3 nm, before a complete drop of intensity at 1.7 nm. In the central region of the water maximum density, the CDD of Nagma corresponding to biomole-cules is lower and linearly increases until 1.1 nm

where it crosses the water profile. Beyond this value the Nagma profile is dominant and it shows three distinct peaks regularly separated by 0.4 nm, and with a progressive increase of intensity and defined shape. This is interpreted as fingerprints of the organization of three shells of highly correlated (packed) hydrophilic molecules, the outer one being the denser and better defined. Few molecules of water are present inside the first two shells, and almost none is found in the last shell which lies at a distance in close proximity to the SWCNT walls. Given the extension of the available inner space within which the water molecules are essentially confined into (0.4 nm), and the low density of peptides, we suggest the presence of a wire like structure, reminiscent of the inner water arrangement in the SWCNT as proposed by Zanotti et al [11]. At larger length scales (1 nm), the wire-like structure begins to evolve into a channel-like structure loosing the contours in its peptide density profile.

Figure 4 reports the mean shell thickness, defined as the distance from the SWCNT axis of all confined atoms averaged over all molecules. This quantity is calculated using the 5 ns production trajectory. Qualitatively, the results confirm segregation of water molecules towards the center of the cylinder in presence of hydrophilic biomolecules which are almost in close proximity to the SWCNT walls.

Figure 4 Mean shell thickness as a function of time, for hydrophobic Nalma (open circles), hydrophilic Nagma (open squares) and related water molecules (full circles, full squares) The comparison between shell thickness highlights the fact that the hydrophilic Nagma segregates water molecules towards the center of the SWCNT.


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Figure 5 displays three-dimensional snapshots taken from the production-run MD trajectories, which visualizes the distinct water molecules distributions in the presence of the hydrophilic Nagma (Fig. 5(a)) and hydrophobic Nalma (Fig. 5(b)) are better visualized.

To improve our knowledge on the confinement properties of the hydrophilic/hydrophobic biomole-cules and their accompanying hydration waters, and also investigate our hypothesis of the confined geometrical arrangements, we examine molecular mobility. It is worth mentioning here that the forcefield we use represents the best compromise between the dual objectives of investigating both structural and dynamical aspects. In fact, it provides a good appro-ximation of the experimental dynamic parameters, which enables reproduction of diffusion coefficients for the hydrophilic and hydrophobic solutes (although slightly overestimating water diffusion). However, despite this small overestimation, the ratio between experimental values of water diffusion in the two solutions is conserved (Table S1 in the ESM). For this reason we are confident that a qualitative and relative analysis of the mobility of confined water can also be extracted and interpreted in a meaningful way. To this

Figure 5 Representation of three dimensional snapshots of SWCNT and confined molecules taken from the final 5 ns production runs. (a) SWCNT confinement of Nagma molecules (green color) and water molecules (orange color) distributed in a correlated water network. (b) SWCNT confinement of Nalma molecules (green color) and water molecules (orange color) randomly distributed and showing possibility of water pockets formation. Water molecules escaping from the SWCNT are visible.

purpose, the total mean square displacement (MSD) was calculated directly from the trajectories and the diffusion coefficients inferred. Figure 6(a) represents the MSD as a function of time for free water, hy-drophilic and hydrophobic molecules and associated confined water molecules. We consider the diffusion coefficient calculated in the time interval up to 1 ns, corresponding to the accessible experimental timescale for dynamics (i.e. using neutron scattering spectroscopy). The contributions from transverse and axial diffusion have been also analyzed and are reported in the ESM.

In accordance with to the high number density (see Fig. 2), the crowding affects MSD of the two biomole-cules exhibits an important slowing down as compared to the behavior of highly concentrated solutions. The hydrophobic biomolecules have a diffusion coefficient (D = 0.0093 × 10–5 cm2/s) which is 35 times smaller than in solutions (Table S1 in the ESM) and more than twice that of the hydrophilic peptide (Table S1 in the ESM, D = 0.049 × 10–5 cm2/s). It is worth noting that, given the distinct structural organization, hydrophilic peptides are more mobile than hydrophobic ones. In both cases, the corresponding water molecules diffuse faster by at least a factor 10. It is straightforward to remark that the water confined within the inner cavity of the SWCNT containing hydrophilic peptides is highly mobile, compared to the hydrophobic case, and its diffusion coefficient (D = 0.5 × 10–5 cm2/s) is closer to that of simulated bulk water (D = 3.25 × 10–5 cm2/s). This behavior suggests the presence of a hydrogen bond network with plasticity similar to that of pure water. Considering the results represented in Fig. 1(b)) and the observed high mobility, it is tempting to conclude that water molecules can exchange “at bulk rate” with external molecules, leaving unchanged the final inner equilibrium unchanged.

Finally to support the previous results we finally performed hydrogen bond (HB) analysis of water– water confined molecules in the presence of both Nalma and Nagma molecules. By using geometrical criteria (defined in the ESM) and introducing a tolerance time, in order to eliminate high frequency contributions, we were able to map the HB lifetime. Figure 6(b) represents the average lifetime of all SWCNT confined hydrogen bonded water–water molecules. The results show a clear evidence of specific


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Figure 6 (a) MSD as a function of time for hydrophobic Nalma confined in SWCNT (open circles), hydrophilic Nagma confined in SWCNT (open squares), and related water molecules (full circles, full squares), compared to non-confined in SWCNT free water. (b) Hydrogen-bond lifetime of water–water confined molecules as a function of tolerance time (ps) for both hydrophilic and hydrophobic biomolecules. The results clearly show that confine-ment of water molecules within the SWCNT from hydrophobic Nalma promote significantly longer water–water hydrogen bond lifetimes.

and distinct differences in behavior of water HB in the presence of hydrophilic/hydrophobic molecules. In agreement with observed changes in the diffusion coefficient, the water molecules confined in presence of hydrophilic molecules are characterized by a more rapid HB dynamics as compared to the hydrophobic case. Water–water interactions are more easily renewed most likely with a new partner given their high mobility. However, in presence of Nalma hydrophobic molecules, the trapped pockets of water molecules do

not seem able to easily renew their HB, which results in significantly longer and more “stable” contacts.

Carbon nanotubes in terms of water transport and their ability to enhance confined water dynamics have been extensively investigated both experimentally and by MD simulations [24–26]. On the other hand our results are not directly comparable, both for the orders of magnitude and general conditions, to the flow of water molecules flow as discussed in these investigations. In fact in our case the effect of SWCNT is “screened” by hydrophilic biomolecules especially owing to the induced crowding effect they induce on them. If the hydrophilic confinement enhances water mobility, hydrophobic encapsulation freezes water molecules trapped by the biological packing. The diffusion coefficient inferred for the hydrophobic case, i.e. 0.04 × 10–5 cm2/s, is almost ten times smaller than what is found in the hydrophilic case. Single water molecules or groups of waters arranged in small pockets do not interact with the external waters in solution but “slowly” escape from the hydrophobic environment inside the SWCNT. As it is shown in Fig. 6, being Nalma molecules are relatively “frozen” within the SWCNT, resulting in water molecules that slowly diffuse within a rigid medium. It is possible to speculate that isolated water molecules slowly move towards the small pockets in order to leave the confinement.

It is natural to compare these results to previously investigated hydration-water dynamics of hydrophilic/ hydrophobic molecules in a geometrical context free of constraint. As a function of hydration level (between 1–7 water/molecule) it was observed that the more hydrophobic the peptide the easier is the onset of water diffusion [13] whereas for highly concentrated solutions the water dynamics around hydrophilic surfaces is faster relative to the hydrophobic scenario [18].

Nevertheless, the amount of water confined, and its distribution within the SWCNT and at the bio- interface, is not equivalent in both investigated cases. The hydrophilic case is likely closer to the solution configuration, while in the hydrophobic case the hydration level can be compared to powders. In the latter case, a crucial difference is that the number of water molecules was carefully selected, and binding sites and HB were built following spontaneous water


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affinity. In this context we introduce a new important parameter, the geometry of the confinement medium, with strong interaction with biomolecules. We show that the amount of water, its distribution and dynamics, is related to the characteristics of the biomolecules, and is a function of the nano-confinement and size of the nano-tube. Thus, it is not surprising that, in presence of the hydrophobic Nalma in SWCNT, highly confined water molecules mostly interact with the bio-interface, and thus show a reduced dynamics. Comparing these results to the case of low hydrated powders [13], we see evidence again that HB extent is a critical parameter in hydration water dynamics [18].

4 Conclusions

The highlight of our work is represented by the surprising self-structural organization of aqueous solvated hydrophilic/hydrophobic small peptides under SWCNT confinement. We show for the first time that hydrophilic molecules confined within SWCNT are more prone to highly structured arrangements compared to confined hydrophobic molecules. We also observe that while the latter promotes dehydration, slowly expelling water molecules from the SWCNT cavity, the hydrophilic confinement reveals a well ordered organization of water molecules and an enhanced water mobility. These results shed light on key parameters important for processes involving long- term storage and conservation of biomolecules in the presence or absence of solvation by water molecules. In this perspective in situ pressure treatment, on the CNT, could be considered in order to recover the hydrated or anhydride molecules.

Acknowledgement

D. R. thanks ARC-Santé and region Rhone-Alpes (France), for financial support with the Nanofold project. D. R. is grateful to Dr. Jose Teixeira (LLB, CNRS France) and Dr. Alessandro Cunsolo for discussions and suggestions. D. R. is grateful to Dr. Scott Brown (Sunovion Pharmaceuticals, USA) for scientific discussion and to have reviewed the manuscript to improve the scientific language. B. A. gratefully acknowledges the computing resources provided on

Blues and Fusion high-performance computing clusters operated by the Laboratory Computing Resource Center at Argonne National Laboratory. Electronic Supplementary Material: Supplementary material (full simulation details and parameter definitions) is available in the online version of this article at http://dx.doi.org/10.1007/s12274-015-0907-7.

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Nano Res.

Table of contents

We show for the first time that hydrophilic molecules undergo to highly structured confinements as compared to hydrophobic ones. We also observe that while the latter promotes dehydration, slowly expelling water molecules from the single-wall carbon nanotubes (SWCNT) cavity, the hydrophilic confinement reveals a well ordered organization of water molecules and enhances water mobility.

Nano Res.

Electronic Supplementary Material

Nano-confinement of biomolecules: Hydrophilic con-finement promotes structural order and enhances themobility of water molecules

Bachir Aoun1 and Daniela Russo2,3 ()

1 Argonne National Laboratory, Chicago, IL 60439, USA 2 CNR-IOM, c/o Institut Laue-Langevin, 38400 Grenoble, France 3 Institut Lumière Matière, Université de Lyon, 69622 Lyon, France Supporting information to DOI 10.1007/s12274-015-0907-7

S1 Full simulation details

S1.1 Forcefield

Various forcefields and parameters such as COMPASS27, cvff, pcff, and CHARMM22, have been tested in order to choose the final setup (Table S1). CHARMM22 all-atom empirical potential forcefield [S1, S2] has better reproduction of both structural and dynamical properties based on experimental results of NAGMA and NALMA solution at equivalent concentrations ratios [S3–S5]. Therefore, MD simulations were performed by using the NAMD package [S6] with all-atom dihedral potential corrected variant CHARMM22/CMAP forcefield [S1, S2] in combination with the three-site water TIP3P model.

Table S1 Diffusion coefficients inferred from the tested forcefield

Forcefield D (10–5 cm2/s) Water in 3M Nagma 3M Nagma Water in 2M Nalma 2M Nalma

Compass27 3.92 0.42 3.86 0.24

cvff 3.34 0.81 3.07 0.45

pcff 4.91 1.4 5.01 1.0

CHARMM22 3.25 0.78 2.7 0.33

Experimental values [S4, S5] 1.1 – 0.75 0.31

The CHARMM22 protein forcefield is parameterized for explicit TIP3P water model interaction with amino

acids compounds, and all its atoms parameters and the atomic partial charges were derived from quantum mechanical chemical calculations of the interactions between model compounds and water.

Address correspondence to [email protected]


Nano Res.

The forcefield used has been tested and found to produce the best compromise in order to achieve the objectives of our structural and dynamical investigation.

S1.2 The initial configurations

Simulations were carried out at 295 K using different concentrations of Nagma and Nalma solutions in the NPT ensemble. The Langevin piston method is used to control the pressure, with an integration time step of 1 fs. Three-dimensional cubic periodic boundary conditions were applied with a long distances interaction cutoff fixed at 12 Å using the Particle Mesh Ewald (PME) method was used for the long-range coulomb forces calculation. The total number of solute molecules (Nagma, Nalma) was fixed at 100 and the numbers of water molecules were adjusted according to the desired concentration (Table S2). The initial configurations were constructed to be as dispersed as possible in order to avoid aggregation. Finally, energy minimization and 5 ns of equilibration were performed and followed by a production run of 1 ns that was used for comparison to experimental data. While the TIP3 water model seems to give a rather high diffusion constant compared to the experiment, the Nalma solute diffusion matches the experimental diffusion coefficient (Table S2).

Table S2 Molecular dynamics parameter of Nagma and Nalma molecules in water solution

CHARMM22 parameters & experimental values

Nalma 1:25 2M Nalma 1:50 1M Nagma 1:19 3M Nagma 1:50 1M

Solute molecules number 100 100 100 100

Water molecules number 2,554 5,000 1,948 5,000

Mean box length (Å) 47.4 56.3 42.4 55.1

Simulation water D (10–5 cm2/s) 2.7 3.5 3.25 3.9

Simulation solute D (10–5 cm2/s) 0.33 0.4 0.78 0.91

Experiment watera D (10–5 cm2/s) 0.75 1.26 1.1 1.65

Experiment solutea D (10–5 cm2/s) 0.31 0.36 – – a Difusion constants derived from Refs. [S4, S5].

However, as the main focus of this work is on the solute behavior, we only qualitatively compare the dynamical

parameters of the water molecules. Figures S1(a) and S1(b) report the atomistic representations of two initial conditions that have been tested in

order to perform simulation on the whole SWCNT and bio-molecule system. Figure S1(a) is build from an equilibrated solution state of Nalma or Nagma which have been previously used to choice the used potential. Adding an empty SWCNT in the box, we noticed that the final equilibration time was extremely long (over 200 ns), because water molecules first fill the SWCNT and then they are replaced from the biomolecules. For this reason a different strategy was used. Figure S1(b) shows the random generated system where water molecules and biomolecules partially fill the SWCNT as initial condition. During the equilibration, the biomolecules immediately load the SWCNT and the total stabilization time was of the order of 100 ns. Therefore, we used this setup for our calculation.

It is important to note that different lengths and diameters of SWCNT have been also tested in order to control the molecules structure dependence from the size of the nanotube. We verified that a cutoff of 1.5 nm enables the filling of the SWCNT with the biomolecules and beyond 2.0 nm above, the structural behavior and do not seems to change. Independence from the CNT length has been also verified up to 10 nm. Consequently, in order to improve the simulation time/performance we decided to use SWCNT of 4 nm diameter and 5 nm long.


Nano Res.

Figure S1 (a) Initial condition builds from the final equilibrated state of bio-molecules in solution, at the desired concentration, and an empty SWCNT. The equilibration state is reached after 200 ns. (b) Initial condition builds from a randomly generated systems containing SWCNT, water molecules and bio-molecule at the desired concentration. The equilibration state is reached in a time range of 100 ns. Both configuration s represent the Nagma molecule case.

S1.3 Structure

In order to compare with the X-ray diffraction structure factor and with previous molecular dynamics simulations [S3] we also calculated the I(Q) X-ray scattering function (Fig. S2). The peaks and shifts positions as a function of solvent concentration agree with the experimental results [S2]. Therefore, increasing the concentration, we observed that the main peak at Q ~1.8 Å–1 of Nalma solution, decrease in intensity and shifts to lower Q values while the bump at Q ~0.8 Å–1 is more pronounced. An equivalent behavior is also observed for the Nagma solution, for what the main diffraction peak is concerned.

Figure S2 Simulated X-ray scattering intensity pattern for Nagma and Nalma as a function of concentration, in water solutions.


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S1.4 Axial and transverse MSDs

In order to confirm the consistency of our results and conclusions we also calculated the axial and transverse diffusion for both Nalma/water and Nagma/water systems. Confined atoms and molecules in SWCNT axial and transversal MSD components, are computed upon the transformed coordinates cntr to the SWCNT principal axes referential system

1 2 3( ) ( , , )P t P P P . As 1P , 2P and 3P are the main, secondary and tertiary

principal axes, atoms inside the SWCNT are striped out of the global SWCNT diffusion by applying the following time dependent transformation matrix MT

1

1, 1, 1,

T 2, 2, 2 ,

3, 3, 3,

( ) ( ) ( )( ) ( ) ( ) ( )

( ) ( ) ( )

x y z

x y z

x y z

P t P t P tt P t P t P t

P t P t P tM And

cntcnt cnt T

znt

( )X x

r Y t yzZ

M

Therefore in the SWCNT principal axes referential system, axial MSD are computed upon cnt( ,0,0)X and transversal MSD upon cnt cnt(0, , )Y Z vectors and the total MSDs is now the sum between the axial and the transverse MSDs.

From the water confinement point of view (Fig. S3(a)) we found that transversal diffusion of water molecules is dominant up to 1 ns while axial diffusion takes over over confirming the presence and the external exchange of the water molecules in the channel. In addition, results reported in Fig. S3(b) show, for Nagma molecules, an important transverse diffusion as compared to the axial diffusion, in the whole observed timeframe.

Figure S3 Axial, transverse and total MSDs calculated for (a) water confined with Nagma molecules; (b) Nagma molecules; (c) water confined with Nalma molecules; (d) Nalma molecules.


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For Nalma and the few trapped water molecules, diffusion is remarkably different compare to Nagma/water confinement. First the few water molecules which have been confined (Fig. S3(c)), essentially experience a local translational diffusion (likely local, considering the magnitude of the MSD) and show a quite negligible axial diffusion. At the same time, Nalma molecules are characteristic of a highly suppressed axial diffusion and a pronounced local transverse diffusion (Fig. S3(d)).

Also in this new calculation, as already noticeable in Fig. 6(a), Nalma/water MSDs are significant lower and with a different time dependence compared to Nagma/water system.

S2 Parameter definitions

S2.1 Number density, ND(t)

It is the ratio between the number of atoms of target molecules found in a defined space by the volume of this space at a specific time t. This calculation is useful to study the evolution in term of quantity of amino-acids compared to water inside the nanotubes. ND(t) is calculated following Eq.(S1)

s1ND( ) ( )t n tV

(S1)

V is the volume of the nanotube, s represents the atoms selection, ns(t) defines the number of atoms found inside the nanotube at time t.

S2.2 Mean shell thickness, MST(t)

It is the average of all the target atoms radii in the nanotube cylinder at time t. This parameter has been used to study how amino-acids compete with water inside the nanotube. MST(t) is calculated using Eq. (S2)

s

s,nt( )s

1MST( ) ( )( ) N t

t d tN t

(S2)

While s refers to the atomic species and nt to the SWCNT, Ns(t) is the total number of target atoms found inside the SWCNT at time t. ds,nt(t) is the distance between the atom s and the SWCNT main axis at time t.

S2.3 Cylindrical density distribution, CDD(d)

Describes the ratio of cylindrical densities of atoms inside the nanotube at a distance d from the nanotube main axis of symmetry. CDD(d) is calculated using Eq. (S3)

s

s( )s

CDD( ) ( )( )2 N t

Vd d d dN t L d d

(S3)

Where Ns(t) is the total number of target atoms found inside the nanotube at time t, V represents the SWCNT volume, ds the distance of the selected atom s from the axis. L is the nanotube length, while Δd is the radial distance tolerance and “ s( )d d d ” is the Kronecker delta that is equal to 1 when “ sd d d ” and equal to zero elsewhere.

S2.4 Mean square displacement, MSD(t)

This parameter gives information about the spatial extent of random motion of the molecules. MSD(t) is


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calculated using Eq. (S4)

2MSD( ) || ( ) (0)||t r t r (S4)

Where “ ” denotes the average over all atoms and ( )r t and (0)r the positions of the atom at time t and 0. When the MSD shows a linear dependence from the time t, the diffusion coefficient D can be calculated from Eq. (S5)

1lim MSD( )6n

D tt

(S5)

The MSD data Fig. 6(a) were calculated directly from the trajectories and from those data we inferred the diffusion coefficient calculated in the time interval up to 1 ns.

S2.5 Hydrogen bond analysis

A hydrogen bond is an attractive force that occurs when an hydrogen is bound to an electronegative atoms. Therefore, it can be defined between exactly three atoms, a donor, the hydrogen and an acceptor. The donor is an electronegative atom (e.g. oxygen, nitrogen, fluorine) with a negative charge, covalently bonded to the hydrogen atom. The acceptor is also an electronegative atom that belongs to the same molecule as the donor and the hydrogen or to a completely different molecule. In this work we investigate water hydrogen bonded dimers confined in carbon nanotube. An oxygen donor atom of a water molecule shares any of the two hydrogen of the same molecule with an acceptor oxygen atom of a near water molecule. In our computation, we consider the following criteria to decide whether an HB is formed: hydrogen-acceptor bond length (BL) < 2.2 Å. and 130° < donor-hydrogen-acceptor bond angle (BA) < 180°. In addition, we also considered a tolerance time factor in order to eliminate high frequency bonds break and re-formation. In this context when the criteria are not satisfy, the hydrogen bond is computed as broken only if the defined tolerance time factor expires before both conditions BL and BA become true again.

References

[S1] MacKerell, A. D. Jr.; Bashford, D.; Bellott, M.; Dunbrack, R. L. Jr.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 1998, 102, 3586–3616.

[S2] MacKerell, A. D. Jr.; Feig, M.; Brooks, C. L. Extending the treatment of backbone energetics in protein force fields: Limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 2004, 25, 1400–1415.

[S3] Hura, G.; Sorenson, J. M.; Glaeser, R. M.; Head-Gordon, T. Solution X-ray scattering as a probe of hydration-dependent structuring of aqueous solutions. Perspect. Drug Discov. Design 1999, 17, 97–118.

[S4] Russo, D.; Murarka, R. K.; Copley, J. R. D.; Head-Gordon, T. Molecular view of water dynamics near model peptides. J. Phys. Chem. B 2005, 109, 12966–12975.

[S5] Russo, D.; Hura, G.; Head-Gordon, T. Hydration dynamics near a model protein surface. Biophys. J. 2004, 86, 1852–1862. [S6] Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kalé, L.; Schulten, K. Scalable

molecular dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781–1802.

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