Interplay between Nanoparticle Wrapping and Clustering of Inner

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Interplay Between Nanoparticle Wrapping and Clustering of Inner Anchored Membrane Proteins Tongtao Yue, Shixin Li, Yan Xu, Xianren Zhang, and Fang Huang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08667 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 12, 2016

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The Journal of Physical Chemistry

Interplay between Nanoparticle Wrapping and Clustering of Inner Anchored Membrane Proteins†

TongtaoYue*,†,‡ , Shixin Li†,‡, Yan Xu‡, Xianren Zhang§, and Fang Huang*,†,‡

†

State Key Laboratory of Heavy Oil Processing, ‡Center for Bioengineering and

Biotechnology, China University of Petroleum (East China), Qingdao, 266580, China. §

State Key Laboratory of Organic-Inorganic Composites, Beijing University of

Chemical Technology, Beijing, 100029, China.

ACS Paragon Plus Environment


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ABSTRACT The receptor mediated endocytosis of nanoparticles (NPs) is known to be size and shape dependent but regulated by membrane properties, like tension, rigidity and especially membrane proteins. Compared with transmembrane receptors which directly bind ligands coated on NPs to provide the driving force for passive endocytosis, the hidden role of inner anchored membrane proteins (IAMPs), however, has been grossly neglected. Here, by applying the N-varied dissipative particle dynamics (DPD) techniques, we present the first simulation study on the interplay between wrapping of NPs and clustering of IAMPs. Our results suggest that the wrapping dynamics of NPs can be regulated by clustering of IAMPs, but in a competitive way. In the early stage, the dispersed IAMPs rigidify the membrane and thus restrain NP wrapping by increasing the membrane bending energy. However, once the clustering completes, the rigidifying effect is reduced. Interestingly, the clustering of longer IAMPs can sense NP wrapping. They are found to locate preferentially at the boundary region of NP wrapping. More importantly, the adjacent IAMP clustering produces a late membrane monolayer protrusion, which finally wraps the NP from the top side. Our findings regarding the competitive effects of IAMP clustering on NP wrapping facilitate the molecular understanding of endocytosis and establish fundamental principles for design of NPs for widespread biomedical applications.


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1. INTRODUCTION In recent years, nanoparticles (NPs) have attracted much global interest due to their potential applications ranging from drug delivery1, 2 to biosensing3 and bioimaging.4 Nearly all their biomedical applications rely on the ability to precisely control their fate once inside the body to achieve the desired effect without adverse consequences.5 To resolve this challenge, it is essential to understand the interaction between NPs and a variety of bio-molecules.6 In general, NPs need to translocate across or be wrapped by cell membranes to accomplish their internalization. Both pathways are dependent on the NP properties, like size,7-11 shape12-14 and surface property,15-19 but also regulated by membrane properties, like tension,20 rigidity,21 curvature22 and transmembrane asymmetry.23, 24 Other factors, like passive and active NP rotation, are found to play essential roles in the kinetic behaviors of NP-membrane interaction.25-27 However, studies in the field of endocytosis often underestimate the role of membrane proteins. This is of undoubted importance because most membrane responses to NPs are the receptor mediated.20, 28, 29 Besides, compared with the transmembrane receptors which directly bind NPs to mediate endocytosis, the indirect role of inner anchored membrane proteins (IAMPs) still remains poorly elucidated. In fact, the complexity of cell membranes is reflected in the abundance of IAMPs, with different properties that depend on the hydrophobic effect and electrostatic interactions with lipid molecules. For example, BAR domains bend cell membranes by anchoring the amphipathic α-helix into the inner leaflet.30 Conversely, curved membranes can recruit amphipathic helices to anchor into it.31 Other important IAMPs, like H2 synthase,32 fatty acid amide hydrolase,33 and microsomal cytochrome P450,34 have also been experimentally determined, revealing the truly complexity of cell membranes. Generally, IAMPs can modulate NP wrapping indirectly in different possible ways. For example, the recruitment of IAMPs may rigidify the membrane and thus restrains its bending to wrap the adhering NPs. In some other cases, contrarily, clustering of IAMPs can produce membrane curvature which may provide



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the caveolae to comfort the internalizing NPs.35 Our previous simulations demonstrated that the clustering of IAMPs can also sense the membrane curvature, and the way they respond to local curvature depends on the hydrophobic length.36 More importantly, the clustering of IAMPs can activate both clathrin assembly and actin polymerization, which generate membrane curvature and membrane tension to collectively promote the NP wrapping.37-40 According to above facts, we believe that the effect of IAMPs on NP wrapping is quite complex and undoubtedly competitive. Particularly, the underlying molecular mechanism of dynamic interplay between NP wrapping and IAMP clustering has never been elucidated by simulations. In this paper, we present the first simulation study on kinetics behaviors of NP wrapping cooperating with clustering of IAMPs. By varying the hydrophobic length of IAMPs, we found that shorter IAMPs display less efficient clustering and distribute more randomely in membrane. The dispersed state was found to rigidify the membrane and thus restrain NP wrapping by increasing the membrane bending energy. For longer IAMPs, they formed larger clusters before completion of NP wrapping. Interestingly, the rigidifying effect was reduced by forming larger clusters. Moreover, an obvious directional diffusion of IAMP clusters towards the boundary wrapping region was identified. At late wrapping stage, the boundary IAMP clustering initiates a membrane monolayer protrusion, which finally wraps the NP from the top side.

2. MODELS AND SIMULATION METHODS 2.1. Mesoscopic Models The coarse-grained models of different components in our simulations are shown in Figure 1. A model lipid molecule was constructed by connecting a headgroup with three hydrophilic beads (H) to two tails, each containing five hydrophobic beads (T).41 The IAMPs were built by linking a bundle of Np chains together with springs to form a relatively rigid body, in which each chain was built by connecting 3 hydrophilic (PH) and ntp hydrophobic beads (PT).36, 42 In this work we set Np = 7, and the number of hydrophobic beads for each chain (ntp) was varied from 3 to 7 to consider its influence on clustering and interplay with NP wrapping. Accordingly, the diameter of IAMPs


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was 1.4 nm and the length was varied from 2.8 nm to 4.6 nm. To model the specific receptor-ligand interaction, 50% of the lipids in the memrbane were set to behave as receptors (R), which interact with ligands on the NP surface.43,

44

Each NP was

constructed by arranging the hydrophilic beads and was constrained to move as a rigid body. Water molecules, which are modeled as single beads (W) and other components were not allowed to enter the interior of the NPs. The ligands coated on the NP surface were modeled as single solid beads (L) and were set to interact with the hydrophilic headgroup of each receptor.

2.2. DPD Method Full technical details of the simulation method are presented in the Supporting Information. In brief, the mesoscopic coarse-grained method of dissipative particle dynamics (DPD) was used in this study. Although DPD was originally developed to simulate the hydrodynamic behavior of complex fluids,45-47 it has been successfully used to reproduce the the phase behaviors of lipid membranes,48, 49 and to explore the interaction between NPs and membranes.50-57 In DPD, the dynamics of each element unit is governed by Newton’s equation of motion, dri/dt = vi and dvi/dt = fi/mi, similar to the molecular dynamics method. The total force exerted on bead i by bead j includes conservative, dissipative, and random force. The interaction parameters between beads of same type were set to aWW = aHH = 25 and aTT = 15, and those between the different types of beads were aTW = 80, aHT = 50 and aHW = 25 (see ESI Methods for details of the interaction parameters). To represent the ligand-receptor interaction, the ligands (L) show an effective attractive interaction with headgroups of membrane receptors (RH), and the interaction parameter aLRH was thus set to 4.0. As initial configuration of the simulations, a ligand coated NP was positioned in close to proximity above the surface of a membrane (Figure 1A). The size of our simulation box was 80×80×50 rc3, and the periodic boundary condition in all directions was taken into account. A number of IAMPs was uniformly inserted in the



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inner leaflet. The number density of the simulation box was fixed to 3. The reduced DPD units can be converted to SI units by mapping the membrane thickness and lipid diffusion coefficient. In experiments, the thickness of a lipid bilayer is of the order of 5 nm and the in-plane diffusion constant of lipids is about 5.0 µm2/s. Accordingly, one DPD length unit corresponds to approximately 0.65 nm in physical units and the time unit corresponds to 16 ps. All simulations were performed in N-varied VT ensembles, in which the targeted membraen surface tension can be controlled by monitoring the lipid number per area ( ρ LNPA ) in the membrane boundary region (see ESI Methods for details of the N-varied DPD method).36 Unless specified, in our simulations the value of ρ LNPA was set to 1.68 to represent the weak negative membrane surface tension.31

3. RESULTS AND DISCUSSION Rational design of NPs for drug delivery requires our thorough understanding of interaction between NPs and membranes. In particular, the receptor-mediated endocytosis is found to be the major internalization mechanism for ligand coated NPs, where the receptor-ligand binding competes with membrane bending to drive this passive endocytosis. However, thorough understanding of this progress has been hindered by knowing the truly complexity of membrane and especially a variety of membrane proteins. Compared with transmembrane proteins which directly contact with NPs to play role, the influence of IAMPs is hidden and thus easily underestimated. To understand the role of IAMPs on NP wrapping generally requires direct measurements in a single cell and is thus very difficult and challenging. Computer simulation, on the contrary, can provide some useful insights into the molecular mechanism of this problem. Our simulations are thus motivated by above facts and consideration, aiming to elucidate the dynamic interplay between NP wrapping and IAMP clustering.

3.1. NP wrapping regulated by IAMP clustering To better understand the effect of IAMP clustering on NP wrapping, we first simulated


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NP wrapping in the absence of IAMPs. Three NP diameters, including 5.2 nm, 7.8 nm and 10.4 nm, were used. We provide in Figure 2 the typical snapshots (A-C), time evolution of wrapping percentage (D) and that of NP position along z direction (E). Here, the wrapping percentage is defined as the ratio of ligands that contact with membrane. Apparently, both rate and pathway of NP wrapping are dependent on the NP size. While larger NPs were finally wrapped by membrane completely, smaller NPs underwent incomplete wrapping under current conditions (Figure 2A-C). More importantly, the whole wrapping process can be roughly divided into four stages. They are NP adhesion stage, membrane bending and wrapping stage, membrane monolayer protruding stage and tuning stage. The first stage is featured with a sudden increase of wrapping percentage (Figure 2D). In this stage, no obvious membrane deformation was observed and the rapid increase of wrapping percentage was thus attributed to the fast NP adhesion on the membrane surface. After the adhesion completed, we found that smaller NPs had higher wrapping percentage, while the wrapping of larger NPs lagged behined. We focus on the molecular architecture of lipids around the NP. It appears that the headgroups of adjacent receptors extended upwards to bind ligands on the NP surface (Figure S1). Under the same extending range, smaller NPs could reach a higher ligand ratio that contact with the receptors. In the stage of membrane bending, the higher extent of wrapping for smaller NPs was gradually overtaken by that of larger NPs. In this stage, higher extent of NP wrapping is a competition between NP-membrane adhesion energy and membrane bending energy. For larger NPs, both higher adhesion area and reduced curvature promote its wrapping by membranes. Once the wrapping percentage exceeded a critical value, it came to the next stage, in which the upper leaflet at the wrapping front started to protrude and wrapped the NP from the top side, whereas the lower leaflet bending was slowed down. To reduce the bending energy for the lower leaflet, the wrapped NP gradually moved upwards in the last tuning stage (Figure 2E). Next, we introduced 100 IAMPs to consider clustering and probe its effect on NP wrapping. Considering that longer IAMPs were proved to display higher extent of clustering,58 we first set ntp = 7 for all IAMPs. The time evolutions of wrapping



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percentage with and without IAMPs are presented in Figure 3. Interestingly, both rate and pathway of NP wrapping were affected by IAMPs, but in contrary ways in early and late wrapping stages. In the early adhesion and especially membrane bending stage, the presence of IAMPs apparently hindered NP wrapping. The restrained wrapping was reflected by the slowed increase of wrapping percentage compared with that without IAMPs (see the black arrow in Figure 3). However, once the wrapping came to the late stage, the NP wrapping was contrarily promoted by IAMPs. In more details, the onset of membrane monolayer protrusion, which finally accomplished the NP wrapping, appeared earlier than that without IAMPs (see the red arrow in Figure 3). Specially, for smaller NP with diameter of 5.2 nm, the incomplete wrapping without IAMPs (Figure 2A) finally developed into full wrapping in the presence of IAMPs. We analyze that the stepwise and reverse modulation of IAMPs on NP wrapping is determined by the complex competition between speed of NP wrapping and that of IAMP clustering. Once the former dominates, the relatively dispersed IAMPs will restrian NP wrapping. Otherwise, the aggregated IAMPs turns to promote NP wrapping. To test our speculation, we further studied the NP wrapping in the presence of IAMPs with different hydrophobic lengths of ntp = 3 and ntp = 7, respectively. The NP diameter was set to 5.2 nm because no complete wrapping was observed in the absence of IAMPs (Figure 2). In Figure S2 we provide the evolutions of cluster number of IAMPs to simply probe the effect of hydrophobic length on clustering behaviors. As expected, both rate and extent of clustering depend on the hydrophobic length. In our previous study we have demonstrated that the IAMP clustering is due to the membrane-mediated interaction, and only when the insertion depth exceeds half the membrane thickness, the strong clustering of IAMPs appears.36 Note that the comparison with our previous work is only qualitative since the different IAMP density and especially the existence of NP wrapping. The detailed wrapping pathway and evolutions of wrapping percentage are given in Figure 4. Apparently, the modulation of NP wrapping by clustering of IAMPs is dependent on their hydrophobic length. If the hydrophobic length of IAMPs is shorter


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than half the membrane thickness (ntp = 3), no complete wrapping was finally observed (Figure 4A). For longer IAMPs, full wrapping was achieved via membrane monolayer protrusion, which was mediated by the boundary IAMP clustering. From bottom view of the typical snapshots, we found that shorter IAMPs (ntp = 3) disperse randomely in membrane (Figure 4A), while longer IAMPs (ntp = 7) form larger clusters (Figure 4B). By comparing the dynamics of both IAMP clustering (Figure S2) and NP wrapping (Figure 4C), we found that shorter IAMPs form clusters much slower than NP wrapping, while the clustering of longer IAMPs completes before completion of NP wrapping. To energetically probe how IAMPs affect NP wrapping, we calculated approximately the membrane bending rigidity with and without IAMPs. The calculation is based on a simple relation in which the bending rigidity is expressed in terms of the area compressibility and the membrane thickness.59 First, we calculated the membrane tension as functions of lipid area. Here, for each lipid area, we calculated three values of surface tension. For comparison, we first calculated the tension for a membrane without IAMPs. In the presence of IAMPs, we further distinguished two states of IAMPs. They are the dispersion state and the aggregation state, respectively. Considering that in our simulations longer IAMPs (ntp = 7) form stable clusters at about t = 2.0 µs (Figure S2), we calculated the two values of tension by averaging them seperately from the first 1.0 µs and last 1.0 µs simulation. As shown in Figure S3, one finds that the tension changes its sign at a certain value of lipid area, A0. For lipid areas A close to A0, one finds a Hookian behavior and the tension behaves as γ ≈ K A ( A − A0 ) / A0 , which defines the area compressibility modulus KA. Then the membrane bending rigidity is given by the simple relation 2 κ = K Alme / 48 , where lme is the membrane thickness. Finally, the calcualted membrane

bending rigidities are given in Figure 4D. Apparently, the membrane bending rigidity is higher in the presence of IAMPs, and is further increased by increasing the IAMP density. Interestingly, once IAMPs form stable clusters, the bending rigidity is contrarily decreased, but still larger than that of membrane in the absence of IAMPs.



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Our calculations of the membrane bending rigidities under different conditions coincide with above simulations which indicated that both nature and extent of influence of IAMPs on NP wrapping is determined by competition between rate of IAMP clustering and that of NP wrapping. In the early membrane bending stage, the dispersed IAMPs restrain NP wrapping by increasing the memrbane bending rigidity. As the clustering proceeds, however, the rigidifying effect is gradually reduced. Besides, for longer IAMPs, the formed clusters preferentially locate at the boundary wrapping region, while no IAMPs were found beneath the NP wrapping (Figure 4B). The heterogeneous distribution of IAMP clusters thus made the restraining effect on NP wrapping further reduced. Besides the reduced rigidifying effect, in the late wrapping stage, the IAMP clusters around NP wrapping accelerate the protruding of membrane monolayer which finally wraps the NP from the top side. We would like to note that transmembrane receptors also increase the membrane bending rigidy in a similar way. The direct binding between receptors and ligands thus competes with the increased membrane bending rigidity to mediate the complex membrane response. Recently, Zhang et al. applied the similar model to systematically investigate the influence of both structure and length of receptors on the NP wrapping by a lipid membrane.29 Under the complex competition between receptor clustering, increased membrane bending rigidity and receptor-ligand interaction, a diverse membrane responses were identified, including membrane adhesion, partial NP wrapping, full NP wrapping and membrane rupture. For IAMPs, the situation is more complex. Besides the asymetric insertion which may induce a non-zero spontaneous curvature, their distribution in membrane is indirectly affected by the NP wrapping. This is different from transmembrane proteins which bind NPs to directly regulate their clustering and membrane distribution.

3.2. IAMP clustering senses NP wrapping As discussed above, both extent and distribution of IAMP clustering in membrane collectively determine in which way it influences the NP wrapping. While shorter IAMPs disperse randomly in membrane and restrain NP wrapping by increasing the


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membrane bending rigidity, longer IAMPs rapidly form clusters to reduce this effect. Moreover, the boundary IAMP clusters facilitate the membrane monolayer protrusion, which finally accomplish the NP wrapping. In this section, we probe how IAMP clustering displays heterogeneous distribution in membrane with NP wrapping. First, to characterize the preferential location of IAMP clustering, we distinguish two local regions corresponding the NP wrapping site. They are the boundary wrapping region and the bottom wrapping region, respectively (Figure 5A). Then we seperately calculated the local IAMP densities in these two regions. Considering that short IAMPs display weak clustering and random membrane distribution, here we set the IAMP length to ntp = 7. As shown in Figure 5B, for smaller NP with a diameter of 5.2 nm, the local IAMP density in the boundary wrapping region shortly increased to a higher value, while that in the bottom region accordingly decreased to near zero. For larger NP with a diameter of 7.8 nm, the trend is similar but less obvious due to larger area of the bottom wrapping region (Figure 5C). This implies that longer IAMPs form larger clusters and preferentially locate at the boundary region of NP wrapping. To directly visualize how IAMPs prefer the boundary region of NP wrapping, we chose four typical IAMPs and tracked their trajectories in the plane of membrane. For smaller NP (D = 5.2 nm), all four probe IAMPs finally diffused directionally from different initial positions to the boundary region of NP wrapping (Figure 6A). As we increased the NP diameter to 7.8 nm, however, four probe IAMPs displayed different diffusive behaviors, depending on their initial positions (Figure 6B). For the IAMP initially locating at the boundary wrapping region, it seemed to be trapped by NP wrapping and no apparent diffusion was observed during the simulation. For the IAMP initially locating at the bottom wrapping region, suprisingly, no apparent migration from bottom to boundary region was observed. For the other two IAMPs initially locating at the periphery of the boundary region, they were found to finally diffuse to the boundary region. To confirm further the preferential location of IAMP clustering at the boundary wrapping region, we initially inserted four IAMP clusters, each having nine IAMPs, at the boundary region of NP wrapping. Three hydrophobic lengths of IAMPs, including



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ntp = 3, 5, and 7, were used to explore their effect on clustering and diffusive behaviors. Both typical snapshots and evolutions of local IAMP density are given in Figure 7. It appears that both clustering stability and diffusivity are strongly dependent on the hydrophobic length of IAMPs. Shorter proteins were found to disaggregate, while longer proteins kept clustering during the simulation. More importantly, IAMPs with different hydrophobic lengths display different ability to sense the boundary wrapping region. For shorter IAMPs, they were found to disperse more randomly in both bottom and boundary wrapping region, while for longer IAMPs, they are quite stable clustering at the boundary region and no IAMPs were found to move out of the region to either bottom or peripheral region. Next, we calculated the free energy change along distance between one IAMP and NP center to energetically analyze its preferential location in membrane with NP wrapping (Figure 8, see ESI Methods for details of the free energy calculation).15, 23 To generate different initial configurations, we artificially inserted the IAMP in membrane with one fully wrapped NP. Here, the NP diameter was set to D = 11.6 nm. We first consider one long IAMP with hydrophobic length of ntp = 7. Apparently, before entering into the boundary region, the free energy keeps nearly unchanged, indicating the random membrane distribution of IAMPs in the absence of NP wrapping. As the IAMP gradually approaches the wrapped NP, a striking decrease of free energy was observed. Further approach contrarily leads to a free energy increase, until the IAMP enters ino the bottom region. This accounts for the prefered location of IAMPs at the boundary wrapping region. According to the typical snapshots, the IAMP locating at d = 9 nm is well stabilized by the membrane monolayer protrusion, while at both bottom and distant region, an apparent membrane perturbation is induced by positive hydrophobic mismatch between protein length and membrane thickness. For comparison, we also calculated the free energy change for shorter IAMP (ntp = 3). As expected, the free energy curve is less evident, which coincides with the randomely dispersed state in membrane. While the clustering dynamics of IAMPs has been well elucidated (Figure S2),42, 58, 60

the sensing behaviors of IAMP clustering to NP wrapping are apparently of equal


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importance. Now, we discuss in-depth why IAMP clustering displays heterogeneous distribution in membrane with NP wrapping (Figure 9). In our previous study, we have shown that the clustering mechanism of intrinsic anchored proteins is ascribed to the local membrane perturbation, which generates long-range protein-protein interactions.36 In the presence of NP wrapping, however, the situation is much more complex, because both ultrastructure and mechanics of membranes are reconstructed by the adsorbed NPs. Specifically, one apparent phenomenon is that the NP wrapping generates membrane curvature. From bottom view, the region beneath the wrapping site has a positive curvature, while that of the boundary region is negative. In more detail, once the NP starts to be wrapped by membrane, the receptors on the membrane diffuse to the wrapping site and bind strongly with ligands on the NP surface. At the front of NP wrapping, the headgroups of adjacent receptors extend upwards to bind more ligands. The lipid extension further generates a slight protrusion,61 the growth of which subsequently wraps the NP from the top side. To both stabilize the protrusion, and, more importantly, reduce the membrane perturbation, adjacent IAMP clusters directionally diffuse to the boundary region of NP wrapping. Furthermore, it facilitates the monolayer protruding which finally accomplishes the NP wrapping. For both lipids and receptors that beneath the NP, we note that their mobility is strongly restrained. They thus behave like a gel phase induced by the NP adhesion.62 For IAMPs initially locating at this region, they are dynamically trapped and thus no directional diffusion was observed for long IAMPs initially locating at the bottom region (Figure 6B).

4.

Conclusions

In summary, we present the first simulation studies on the interplay between NP wrapping and IAMP clustering. Our simulations suggest that both rate and pathway of NP wrapping can be regulated by IAMP clustering, but in a competitive way, depending on the hydrophobic length of IAMPs. Shorter IAMPs display weak clustering and disperse randomly in membrane with and without the NP wrapping. The random dispersion of IAMPs was found to rigidify the membrane and thus



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restrains NP wrapping by increasing the membrane bending energy. For longer IAMPs, however, the situation is more complex. Owing to their rapid clustering, the rigidifying effect on membrane is effectively reduced before completion of NP wrapping. Besides, the IAMP clustering was found to sense the NP wrapping. In general, the IAMP clusters prefer to locate at the boundary wrapping region. Such preferential location is to reduce the membrane perturbation by positive hydrophobic mismatch. More importantly, it facilitates the NP wrappinng by promoting the front membrane monolayer protrusion, which subsequently wraps the NP from the top side. These findings help our thorough understanding the endocytosis mechanism, and suggest that the clustering of IAMPs should be taken into account when designing NP-based drug delivery vectors. Future works, besides the fundamental studies highlighted above, includes the further investigation of how detailed architecture of IAMPs senses and regulates the NP wrapping.

ASSOCIATED CONTENT Supporting Information The Supporting Information containing the detailed description of simulation method, interaction parameters, and free energy analysis, local configuration of initial NP adherion on membrane surface without IAMPs, a simple comparison of clustering dynamics of IAMPs with different hydrophobic lengths, and the calculated surface tension as a function of lipid area is available free of charge on the ACS Publications website at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Tel: 86-532-86981135, Fex: 86-532-86981135, E-mail: [email protected] (T. Yue) *Tel: 86-532-86981560, Fex: 86-532-86981560, E-mail: [email protected] (F. Huang) Author Contributions T. Y. and S. L contributed equally to this work. T. Y. and F. H. conceived and designed


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the project. T. Y. designed and S. L. performed the simulations. The manuscript was written by T. Y. and S. L. and revised with Y. X., X. Z. and F. H., with contributions from all authors. Notes The authors declare no competing financial interest.

Acknowledgments This work was financially supported by the National Natural Science foundation of China (No. 21303269 and 21273287) and the Natural Science foundation of Shandong

Province

(ZR2013BQ029).

The

authors

thank

the

National

Supercomputing center in Shenzhen for providing excellent computer time.

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Figure 1. Schematic representation of the DPD models. A: Different components, B: Lipid molecule, C: Receptor molecule, D: Spherical NP with ligands coating on the surface, E: Inner anchored membrane proteins (IAMPs) with hydrophobic lengths of 7, 5 and 3, respectively.



1.0

D

24

E

D = 5.2 nm D = 7.8 nm D = 10.4 nm

22 0.8

20 0.6 0.4

D = 5.2 nm D = 7.8 nm D = 10.4 nm

0.2 0.0 0

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Wrapping percentage

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5000

18 16 14 12 10 0

1000

2000

3000

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5000

Time (ns)

Time (ns)

Figure 2. Size-dependent wrapping of NPs by membranes in the absence of IAMPs. A-C are the typical snapshots of NP wrapping with different sizes (A: 5.2 nm, B: 7.8 nm, C: 10.4 nm); D and E are the time evolutions of wrapping percentage and positions of NPs along the membrane normal direction.


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Wrapping percentage

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1.0

Restraining Promoting

0.8 0.6

D = 5.2 nm with no IAMPs D = 7.8 nm with no IAMPs D = 10.4 nm with no IAMPs D = 5.2 nm with 100 IAMPs D = 7.8 nm with 100 IAMPs D = 10.4 nm with 100 IAMPs

0.4 0.2 0.0 0

1000

2000

3000

4000

5000

Time (ns) Figure 3. Competitive regulation of NP wrapping by IAMP clustering. Three NP diameters, including 5.2 nm, 7.8 nm and 10.4 nm, were used. For each NP size, the evolutions of wrapping percentages both with (solid line) and without IAMPs (semi-transparent) are provided. The hydrophobic length of IAMPs is ntp = 7.



35 1.0

C

34

dispersed state aggregated state

D

33 0.8 0.6 0.4

ntp = 7

0.2

ntp = 3 0.0

0

1000

2000

3000

4000

Time (ns)

5000

Bending rigidity

Wrapping percentage

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32 31 30 29 28 27 26

without IAMP 25 120 140 160 180 200 220 240 260 280 300 320 340

Number of IAMPs

Figure 4. Regulated NP wrapping by IAMP clustering. A and B are the typical snapshots from both side sectional and bottom view; C shows the evolutions of wrapping percentage with IAMPs having different hydrophobic lengths; D shows the calculated membrane bending rigidity with and without IAMPs. The NP diameter was set to 5.2 nm. The membrane monolayer protrusion mediated by IAMP clustering is highlighted by two black arrows.


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0.07 0.06

C

B

0.06

Density of protein

0.05

Density of protein

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0.04

Bottom region Boundary region

0.03 0.02 0.01

0.05 0.04 0.03


0.02

0.00 0

1000

2000

3000

4000

5000

0.01

0

1000

Time (ns)

2000

3000

4000

5000

Time (ns)

Figure 5. Preferential location of IAMP clustering in membrane with NP wrapping. A is the schematic definition of two different wrapping regions: boundary region (yellow) and bottom region (blue). B and C give the evolutions of IAMP densities in these two regions. The NP diameters are set to 5.2 nm (B) and 7.8 nm (C), respectively. The hydrophobic length of IAMPs is ntp = 7.



35

A

35

30

B

30

Y (n m )

Y (n m )

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25

25

20

20

15

15

15

20

25

30

35

15

20

X (nm)

25

30

35

X (nm)

Figure 6. Trajectories of four probe IAMPs with hydrophobic length of 7 initially locating at different positions. The NP diameters were set to 5.2 nm (A) and 7.8 nm (B), respectively. Both boundary wrapping region and bottom wrapping region are labeled by gray and green color. The start and end positions of each probe protein are marked with solid circle and triangle symbols, respectively.


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0.08

Density of IAMPs

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D

F

E

0.06


0.04 0.02


0.00 0

1000

2000

3000

Time (ns)

4000

5000

Bottom region Boundary region 0

1000

2000

3000

4000

5000

Time (ns)

0

1000

2000

3000

4000

5000

Time (ns)

Figure 7. The effect of hydrophobic length of IAMPs on clustering and preferential location in membrane with NP wrapping. A shows typical snapshots and B-D show the evolutions of local IAMP densities at two distinguished regions. The NP diameter was set to 7.8 nm and the hydrophobic lengths are 3 (A and D), 5 (B and E), and 7(C and F), respectively.



2 1

Free energy change

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0 4'

1'

-2

2'

-4 4

2 3'

-6

ntp = 7

-8

ntp = 3

3

-10 8

9

10

11

12

Distance

Figure 8. Free energy change as a function of distance between IAMP and NP center. The diameter of NP was set to 11.6 nm. Two IAMPs with hydrophobic lengths of 3 and 7 were considered for comparison.


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Figure 9. Schematic depiction of interplay between NP wrapping and IAMP clustering.



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445x240mm (96 x 96 DPI)


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Interplay between Nanoparticle Wrapping and Clustering of Inner

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