Maleic Acid Copolymers Form SMALPs by Pulling Lipid

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Styrene-maleic acid copolymers form SMALPs by pulling lipid patches out of the lipid bilayer Philipp S. Orekhov, Marine Bozdaganyan, Natalia Voskoboynikova, Armen Y. Mulkidjanian, Heinz-Juergen Steinhoff, and Konstantin V. Shaitan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03978 • Publication Date (Web): 18 Feb 2019 Downloaded from http://pubs.acs.org on February 18, 2019

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Figure 1. Structural formula of a SMA copolymer fragment with the CG beads schematically mapped onto it. The beads are captioned with their selected MARTINI particle types. 82x86mm (300 x 300 DPI)

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Figure 2. Aggregation of periodic SMA copolymers in solution. A-E: Representative self-assembled morphologies of SMA copolymers (3:1 styrene:MA ratio, MA residues carrying the charge of −1) at different concentrations (A, D, E), SMA copolymers with 2:1 styrene:MA ratio (B) and 3:1−2 SMA copolymers (both carboxyl groups deprotonated) (C). Water and ion beads are omitted for clarity. F-H: Gyration radii of a copolymer cluster (F), end-to-end distance of individual SMA copolymers averaged over all the simulated copolymers (G), and distributions of the number of neighbour copolymers at the minimal distance of 8 Å or less (H) for SMA copolymers differing in styrene/MA ratio, charge, and concentration in aqueous solution. 165x86mm (300 x 300 DPI)


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Figure 3. Interaction of periodic SMA copolymers (2:1 ratio of styrene:MA residues, [SSM]17) with a DMPC bilayer. A: Initial configuration of the system; B: a snapshot upon the absorption of the SMA aggregates to the bilayer; C: the final configuration with a pore formed; D: growth of a membrane pore after 1 μs-long simulation with the polarizable water model, which extended the preceding 5 μs-long simulation with the standard water model. Water and ion beads are omitted for clarity. E: Enlarged view of porous defect of the DMPC bilayer stabilized by SMA copolymers. Water is shown as the blue surface. Hereinafter, phosphate, choline and MA moieties are shown as orange, blue and orange spheres, respectively, styrene rings – as yellow triangles, SMA copolymer backbone and lipids – as white and salmon sticks. 165x146mm (300 x 300 DPI)


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Figure 4. Interaction of SMA copolymers (3:1 ratio of styrene:MA residues, [SSSM]13) with a DMPC bilayer. A: Initial configuration of the system; B: a snapshot upon the absorption of the SMA aggregates to the bilayer; C: the final configuration with a disc-shaped protrusion formed. Water and ion beads are omitted for clarity. 82x142mm (300 x 300 DPI)


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Figure 5. Formation of the disc-shaped protrusion stabilized by periodic SMA copolymers (3:1 ratio of styrene:MA residues, [SSSM]13). A-C: Early steps of emergence of the disc-shaped protrusion. Contacts of the lipid tails with the hydrophobic styrene moieties of a SMA copolymer cluster are shown enlarged in the inset. D: Shape and dimensions of the disc-shaped protrusion at the end of the simulation. Water and ion beads are omitted for clarity. 165x113mm (300 x 300 DPI)


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Figure 6. Interaction of statistical (A-C), length-dispersed (D-F) and short-chain ([SSSM]3) (G-I) 3:1 SMA copolymers with a DMPC bilayer. A, D, G: Snapshots upon the absorption of the SMA copolymer aggregates to the bilayer. B, E, H: Early steps of emergence of the disc-shaped protrusion. C, F, I: Final configurations. 165x99mm (300 x 300 DPI)


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Styrene-maleic acid copolymers form SMALPs by pulling lipid patches out of the lipid bilayer Philipp S. Orekhov 1,2,3,∇*, Marine E. Bozdaganyan 1,4,∇, Natalia Voskoboynikova 5, Armen Y. Mulkidjanian 5,6,7, Heinz-Jürgen Steinhoff 5 and Konstantin V. Shaitan 1 1

Department of Biology, Lomonosov Moscow State University, Moscow, Russia

2

Sechenov University, Moscow, Russia

3

Moscow Institute of Physics and Technology, Dolgoprudny, Russia

4

Federal Research and Clinical Center of Specialized Medical Care and Medical Technologies,

Federal Medical and Biological Agency of Russia, Moscow, Russia 5

Department of Physics, University of Osnabrueck, Osnabrueck, Germany

6

A.N. Belozersky institute of Physico-Chemical Biology, Lomonosov Moscow University,

Russia 7

School of Bioengineering and Bioinformatics, Lomonosov Moscow University, Russia

∇

these authors contributed equally.

* To whom correspondence should be addressed: Russia, 141700, Moscow Region, Dolgoprudny, Institutsky per. 9/7, tel.: +7 968 4633964, fax: +7 495 4084254, e-mail: [email protected].


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Abstract Amphiphilic copolymers composed of styrene and maleic acid (SMA) monomers caused a major methodical breakthrough in the study of membrane proteins. They were found to directly release phospholipids and membrane proteins both from artificial and natural lipid bilayers yielding stable water-soluble discoidal SMA/lipid particles (SMALPs) of uniform size. Although many empirical studies indicate the great potency of SMALPs for membrane protein research, the mechanisms of their formation remain obscure. It is unknown which factors account for the very assembly of SMALPs and govern their uniform size. We have developed a coarse-grained (CG) molecular model of SMA copolymers based on the MARTINI CG force field and used it to probe the behavior of SMA copolymers with varying composition/charge/concentration in solution as well as their interaction with lipid membranes. First, we found that SMA copolymers tend to aggregate in solution into clusters, which could account for the uniform size of SMALPs. Next, MD simulations showed that periodic SMA copolymers with styrene:maleic acid ratios of 2:1 ([SSM]n) and 3:1 ([SSSM]n) differently interacted with lipid bilayers. While clusters of 2:1 SMA copolymers induced membrane poration, the clusters of 3:1 SMA copolymers extracted lipid patches from the membrane yielding SMALP-like structures. Extraction of lipid patches was also observed when we simulated the behavior of 3:1 copolymers with varying lengths and statistical distribution of styrene and MA units. Analysis of MD simulation trajectories and comparison with experimental data indicate that the formation of SMALPs requires copolymer molecules with sufficient number of units made of more than two sequential styrene monomers.


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Introduction Determining protein structures is a key for understanding their molecular mechanisms, which is a prerequisite for development of novel therapies and drugs. Transmembrane proteins (TPs) play crucial role in the cell functioning1. They have hydrophobic regions containing a high fraction of non-polar amino acids and hydrophilic regions with a high proportion of polar amino acids. The complementarity between the hydrophobic regions of proteins and the hydrophobic regions of lipid bilayer, as well as between the hydrophilic regions of proteins and the extracellular and intracellular environments, stabilizes TPs within the bilayer, thus preventing their extracellular and cytoplasmic regions from flipping back and forth2. Moreover, TPs are often additionally stabilized by interactions with particular components of biological membranes3. Hereby, TPs need to be studied in their native lipid environment. By now, various TP extraction methods have been developed that usually were based on using diverse detergents for membrane solubilization45. Recently it was shown that detergents could be avoided when amphiphilic styrene maleic acid (SMA) copolymers were used6,7. Such copolymers enable a detergent-free extraction of lipids and membrane proteins both from artificial and natural lipid bilayers yielding Styrol/Maleic Acid Lipid Particles (SMALPs). Usually, copolymers with styrene:maleic acid (MA) ratio of 2:1 or 3:1 are used for producing SMALPs6–16. SMALPs comprise lipid or lipid/protein discs surrounded and stabilized by a SMA copolymer belt and have a diameter around 10 nm depending on the preparation routine. Within a particular preparation, the size of particles is uniform, which renders them suitable for diverse experimental techniques. Importantly, the SMA copolymer-mediated lipid solubilization is non-selective concerning the lipid type17–19. SMALPs have a number of significant advantages compared to other TP extraction methods: they are easier and cheaper to manufacture than nanodiscs (a membrane system similar to SMALP but surrounded by a scaffolding protein); the SMALP method completely eliminates the need for detergent, ensuring that the local lipid environment around the TP is preserved17,20,21. SMA copolymers were shown to be able to form SMALPs with proteins of different sizes with up to 40 transmembrane helices17,20,22, with various lipid compositions23 and also they are


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capable of lipid exchange with a lipid monolayer24. By now, several protein structures were resolved using SMALPs: photoreactive bacterial reaction centers25, ABC-family proteins15,26, Complex IV of the respiratory chain of mitochondria27, bacteriorhodopsins6,13,28, potassium channels14,29, G-protein coupled receptors (GPCRs)16, light-harvesting complex II17, nucleoside transporter hENT130. In all these studies, the authors observed no changes in protein functioning, which is an evidence that SMALPs almost do not change the properties of cell membranes and transmembrane proteins. SMALPs are prepared in a single step: an amphiphilic copolymer is added to a membrane fraction containing the necessary proteins, a spontaneous interaction of the copolymer with the membrane takes place yielding lipid discs surrounded by copolymer belts that screen hydrophobic lipid tails from the solvent21,31. The appearance of SMALPs can be traced by following the transparency changes of the solution. For instance, in experiments with palmitoyloleoylphosphocholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG) containing vesicles that were titrated with different amounts of 3:1 SMA copolymers the sample became clear at the critical weight ratio of 1:1.25 (POPC/POPG to SMA, respectively)32. The structure of SMALPs was characterized by different methods6,7,23,31. SMALPs that had been derived from DMPC vesicles were found to have a discoidal shape with a diameter of about ~10 nm and a thickness of 4.6 nm, which corresponds to the thickness of pure DMPC bilayer in the fluid phase. The SMA copolymer belt surrounding the lipids was shown to be ~0.9 nm thick31. Phenyl groups of styrene units intercalate the lipid acyl chains, whereas carboxyl groups interact electrostatically with the head groups of lipids7. Several important points still deserve clarification, namely: (1) the mechanism of how SMALPs are formed; (2) the exact amount of SMA copolymers associated with one SMALP, and also whether all of the associated polymer material is involved in the stabilization of the disc; (3) the difference between mechanisms of forming SMALPs by various copolymer types. Coarse-grain (CG) molecular dynamics (MD) simulations enable studying the evolution of large molecular systems up to microsecond timescales owing to the merger of several heavy atoms


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into pseudo-atoms33. Such approaches have proven themselves to be powerful tools for investigation of vesicle formation and fusion, membrane reshaping by proteins, interactions of membranes with polymers and detergents34–36.

Particularly, a recent paper by Xue et al.37

addressed the interaction between a lipid bilayer and SMA copolymers with 2:1 styrene:MA ratio and doubly charged MA. It was shown that SMA copolymers interacted with the bilayer due to the hydrophobic effect and caused solubilization via formation of small, water-filled pores, which further grew leading to membrane disruption. This study provided the first insight into the molecular mechanism of interaction of SMA copolymers with lipid bilayers. However, the complete formation of SMALPs was not observed in simulations, which encourages further research for clarifying the mechanisms of SMALPs formation. In the present study, by means of coarse-grained MD simulations, we assessed the tendency of different types of SMA copolymers to aggregate in water solution and explored their interaction with model lipid bilayers. We observed formation of SMALPs with statistical and periodic 3:1 SMA copolymers and showed that the mechanisms of interaction of SMA copolymers with lipid bilayers could be different depending on the composition and chain length of SMA copolymers.


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Materials and Methods Parametrization of MARTINI Coarse-Grain model for SMA copolymers The CG models of SMA copolymer were parameterized based on the preceding all-atom (AA) simulations. The AA SMA copolymer model consisting of 52 monomers with the 3:1 styrene/maleic acid ratio was built using the OPLS AA force field38. The SMA copolymer was placed into a cubic box with a side of 9.3 nm filled with water (the TIP3P water model39) and an appropriate number of Na+/Cl− ions, which assured the ionic strength of 0.15 M and zero total charge. Then, the AA simulation was carried out in the NPT ensemble for 70 ns using Gromacs 2018.1 (T=303.15 K maintained by the V-rescale algorithm, P=1 bar controlled by ParrinelloRahman barostat; the integration time step was equal to 2 fs; the Verlet cutoff scheme and particle mesh Ewald (PME) were used for the nonbonded interactions with the cutoff value set to 1.2 nm). The chemical structure of SMA copolymer was mapped to the CG representation (Figure 1) by analogy with the mapping used in the standard MARTINI library for the aspartate and phenylalanine amino acid residues and according to prediction of the Auto MARTINI tool40 . The parameters for the bonded interactions were optimized based on the iterative procedure employed before41, which pursued satisfactory overlap (>75%) between bond/angle/dihedral histograms (Figures S1/S2/S3) obtained from the AA simulation and from a series of CG simulations with parameters adjusted in a stepwise manner. The final set of parameters along with the corresponding coordinate files can be found at https://hpc.mipt.ru/zhmurov/lab/. The latest CG model of the dimyristoylphosphatidylcholine lipids (DMPC) was used42.


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Figure 1. Structural formula of a SMA copolymer fragment with the CG beads schematically mapped onto it. The beads are captioned with their selected MARTINI particle types.

Coarse-Grain MD Simulations All CG simulations were performed in Gromacs 2018.1. For each system, energy minimization using the steep descent algorithm was run prior to equilibration simulation in the NPT ensemble maintained by means of the Parrinello−Rahman barostat (time constant = 12.0 ps, compressibility = 3 × 10−4 bar−1 as recommended in43) and the V-rescale thermostat, respectively. The barostat was applied semi-isotropically in the simulations with lipid bilayers and isotropically elsewhere. For simulations with the polarizable water model, the PME method was used for the long-range electrostatics with the relative permittivity of 2.5. In simulations with the standard water model the reaction field approach was used with the relative permittivity of 15. The time step of 20 fs was used for all simulations. The Verlet pair-lists cutoff scheme was used and the neighbor list was updated every 10 steps. Periodic boundary conditions were used in all simulations. The MD simulations of SMA copolymers self-aggregation were run for 4 μs; all the simulations of SMA copolymer:lipid systems were simulated for 5 μs. The performed MD simulations are listed in Table 1 specifying the types and number of SMA copolymers used in different


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simulations. The sequences of all the simulated copolymers are provided in Table S1. The total simulation time exceeded 70 μs. The custom TCL and Python scripts were used for the analysis of simulations. Table 1. The list of all performed coarse-grained MD simulations. W – with the standard water model; PW – with the polarizable water model; DMPC – dimyristoylphosphatidylcholine. No.

Styrene:MA

MA

Number of

Number of

Simulation

Initial box size

SMA

molar ratio,

char

SMA

lipids

length, μs,

(nm)

concentration,

copolymer

ge

copolymers/the

type

replicas

solvent

ir length (in monomers)

1

3:1, periodic −1

12/52

-

4.0 x 1

16, 16, 16

~5 mM, W

2

3:1, periodic −1

24/52

-

4.0 x 1

16, 16, 16

~10 mM, W

3

3:1, periodic −1

48/52

-

4.0 x 1

16, 16, 16

~20 mM, W

4

2:1, periodic −1

12/51

-

4.0 x 1

16, 16, 16

~5 mM, W

5

3:1, periodic −2

12/52

-

4.0 x 1

16, 16, 16

~5 mM, W

6

3:1, periodic −1

12/52

780 DMPC

5.0 x 2

23, 10, 15

~6 mM, W

6c

3:1, periodic −1

12/52

780 DMPC

1.0 x 1

23.6, 10.3, 13.5

Extension of #6, PW

7

2:1, periodic −1

12/51

780 DMPC

5.0 x 2

23, 10, 15

~6 mM,W

7c

2:1, periodic −1

12/51

780 DMPC

1.0 x 1

25.5, 11.1, 11.4

Extension of #7, PW

8

3:1, periodic −1

3/104+3/52+3/

780 DMPC

5.0 x 2

23, 10, 15

W

26+3/13


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3:1,

−1

12/52

780 DMPC

5.0 x 2

23, 10, 15

W

3:1, periodic −1

48/12

780 DMPC

5.0 x 2

23, 10, 15

W

statistical 10

Results and Discussion Development of SMA copolymer models So far, predominantly copolymers with styrene and maleic acid units in a molar ratio of 2:1 or 3:1 and with molecular weight range of 6-10 kDa were used in experimental studies23,44,45. The two carboxyl groups in the high-molecular weight SMA copolymers have different pKa values: the first pKa is close to 5.0, whereas the second pKa is close to 9.046. Only one of the two carboxyl groups is deprotonated under neutral conditions routinely used for membrane solubilization, leading to a total charge of −1 at the maleic acid unit. The exact monomer sequence of SMA copolymers typically used for SMALP formation remains unclear 46,47,48 while its molar mass dispersity is about 2-3 (for commercially available SMA copolymers (Lipodisq®) from Sigma-Aldrich) Building on these facts, in the present study we have developed several models of periodic and statistical SMA copolymers of different length with 2:1 and 3:1 molar styrene:MA ratios, which were aimed to simulate the indicated properties of SMA copolymers while retaining simplicity to allow clear interpretation of MD simulations. First, a CG model of periodic 3:1 SMA copolymer containing 52 monomers with alternating styrene triads and MA units (carrying each the total charge of −1), [SSSM]13, was created (MW=5.8 kDa) based on the auxiliary all-atom simulation (see Methods for details). The quality of the model was assessed by comparing the end-to-end distance and the gyration radius of the all-atom and coarse-grained SMA copolymers (Figure S4). Then, the optimized parameters from the CG model of the periodic 3:1 SMA copolymers were further used to build CG models of periodic 3:1 SMA copolymers with varying chain length (consisting of 12, 13, 26, 52 and 104 monomers), the statistical 3:1 SMA copolymers and the periodic 2:1 SMA copolymers (with alternating styrene dyads and MA units, i.e. [SSM]17). Sequences of statistical 3:1 SMA copolymers were generated


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according to the penultimate unit model described in 49,50 considering that MA monomers do not homopolymerize. The distribution of styrene dyads, triads, etc. in the generated sequences is shown in Fig. S5. The full list of copolymers used in the subsequent simulations along with their molecular weights and monomer sequences can be found in Tables 1 and S1.

Behaviour of SMA copolymers in solution SMA copolymers are amphiphilic, polyanionic molecules and their solution behavior and ability to solubilize membranes strongly depends on pH value, which affects the protonation states of MA residues and, thus, the total charge of copolymers9,46. We estimated the tendency of SMA copolymers to collapse and aggregate in water from a series of MD simulations (Figure 2), in which we varied the concentrations of the periodic 3:1 SMA copolymer variant (5, 10 and 20 mM, see Table 1, simulations no. 1-5). In addition, we checked the influence of the total charge by running MD simulations with the periodic 2:1 SMA copolymer variant (i.e., with a higher charge density) and the periodic 3:1 SMA copolymer variant with both carboxyl groups of MA deprotonated (i.e., with a −2 net charge at each maleic acid unit, hereafter shortened as 3:1-2 SMA copolymer). We have found out that already at a relatively low concentration of 5 mM (corresponding to 12 copolymers in the simulation box), the SMA copolymers, which were initially randomly spread in the cubic water box (with the edges equaling 16 nm), clustered regardless of the SMA copolymer charge (Figure 2). However, the sizes of these clusters differed significantly; the 3:1 SMA copolymer with one carboxyl per maleic acid deprotonated made the most compact clusters, whereas the 2:1 variant and 3:1-2 SMA copolymers made larger clusters as indicated by the gyration radii (Figure 2F). Evidently, the increase in the net molecular charge within a series of these SMA copolymers (−13, −17 and −26 per SMA molecule, respectively) destabilized the clusters of SMA copolymers and altered the compactness of their hydrophobic cores, which were formed of styrene units, as evident from the corresponding radial distribution functions (Figure S6); the compactness of hydrophobic cores was shown to be essential for efficient insertion of SMA copolymers into lipid membranes46. At the same time, we found out that the causes of the observed less compact conformations of SMA copolymers clusters were different in case of 2:1 and 3:1-2 SMA copolymers. In 3:1-2 SMA copolymers, the end-to-end distances of individual


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copolymers (Figure 2G) became larger indicating their relative extension (Fig. 2G): In 2:1 SMA copolymers the end-to-end distances were similar to that in 3:1 copolymers, which implies that individual 2:1 SMA copolymers remain folded and destabilization of SMA copolymer clusters owed to electrostatic repulsion of individual polymers. With the increase in the concentration of SMA copolymers, the morphology of their selfassembled clusters demonstrated notable changes. Instead of a single cluster, copolymer molecules formed a spread heterogeneous web consisting of a few clusters connected by thin stretches of copolymers (Figure 2D-E, S7), which appears to be a general property of amphiphilic block copolymers51. Also, the individual copolymers became more stretched (Figure 2G). We compared the structures of these cluster aggregates with those of clusters and their aggregates formed at lower concentrations of SMA copolymers by estimating the distributions of an average number of neighboring copolymers for each SMA copolymer molecule (Figure 2G). The calculated distributions evidence the essentially constant intrinsic organization of copolymer clusters. In all of the performed simulations, the SMA copolymers had, on average, 10-12 neighbors within 8 Å distance regardless of the total number of copolymers in the simulation box (Figure 2F).

Figure 2. Aggregation of periodic SMA copolymers in solution. A-E: Representative selfassembled morphologies of SMA copolymers (3:1 styrene:MA ratio, MA residues carrying the


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charge of −1) at different concentrations (A, D, E), SMA copolymers with 2:1 styrene:MA ratio (B) and 3:1-2 SMA copolymers (both carboxyl groups deprotonated) (C). Water and ion beads are omitted for clarity. F-H: Gyration radii of a copolymer cluster (F), end-to-end distance of individual SMA copolymers averaged over all the simulated copolymers (G), and distributions of the number of neighbour copolymers at the minimal distance of 8 Å or less (H) for SMA copolymers differing in styrene/MA ratio, charge, and concentration in aqueous solution.

Interaction between SMA copolymers and lipid bilayers In order to investigate the interaction of lipid bilayers with SMA copolymers with varying styrene:MA molar ratio, chain length and sequences, we started from similar setups placing an appropriate number of SMA copolymers (see Table 1, simulations no. 6-10) at approximately 2 nm from the bilayer consisting of 780 DMPC lipids (Figures 3A and 4A). DMPC was taken for MD simulations as a typical model lipid; it had been used earlier for SMALPs preparation31. Also, this lipid has relatively short acyl chains (14 carbon atoms), which facilitates membrane solubilization23 and thus makes it observable at the timescale of our CG simulations. In the beginning of the simulations, SMA copolymers were in extended conformation but during the first few tens of nanoseconds they became compact (Figure S8) and started to cluster for all the simulated SMA copolymer variants. Interaction of periodic 2:1 SMA copolymers with DMPC bilayers. In case of uni-length periodic SMA copolymers with 2:1 ratio of styrene:MA, [SSM]17, the clusters of copolymers rapidly adsorbed onto the membrane and started to penetrate into it in such a way that after 500 ns of simulation all of the SMA copolymers were inside the membrane (Figure 3B). Upon insertion, the copolymers almost completely disaggregated forming only transient contacts between each other. While initially all the SMA copolymers became adsorbed onto one leaflet, during the course of simulation some of them were also able to cross the membrane (Figure 3B, C). Penetration of SMA copolymers inside the membrane caused several membrane perturbations including distortions of its thickness (Figure S9), bending and formation of porous defects


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(Figure 3C). The latter appeared several times in each of the two replica simulations, which we have carried out to achieve better statistics. These defects were piercing the whole membrane and were lined mostly by the MA residues, while the styrene groups were facing towards the lipid tails. The head groups of some lipids also contributed to the formation of the hydrophilic coating. Altogether, maleic acid residues of SMA copolymers and hydrophilic moieties of lipids supported continuous water pore crossing from one side of membrane to the other (Fig. 3E). The standard MARTINI model provides great performance but it can encounter problems when modeling the behaviour of charged and polar species at the interfaces of water with low dielectric media, such as membranes. To overcome this limitation, we replaced the standard water model by the polarizable one, which has proved itself useful for accurate modeling of membrane electroporation and transmembrane translocation of charged species52. Then, a successive simulation was run for an additional 1 μs. In this simulation, the small pores, which were emerging in the MD simulations with the standard water model, started to grow further destabilizing the membrane (Figure 3D). Apparently, this process would have led to the complete solubilization of membrane, which we did not observe because of the limited size of the simulation box. Interaction of periodic 3:1 SMA copolymers with DMPC bilayers. In case of periodic 3:1 SMA copolymers of similar length, [SSSM]13, the clusters of copolymers also adsorbed onto the surface of bilayer at the early step of the MD simulations but they did not disaggregate in contrast to the 2:1 SMA copolymer variant (Figure 4). Instead, the clusters perturbed the bilayer eventually forming hydrophobic–hydrophobic contacts between the lipid tails and the hydrophobic moieties of SMA copolymers (Figure 5). The amount of these hydrophobic contacts was larger as compared to the simulations with periodic 2:1 SMA copolymers (Figure S10). Within less than 100 ns, the initial hydrophobic defect at the surface of bilayer gave rise to a large protrusion stabilized by a single layer of a SMA copolymer comprising of 377 monomers that corresponds to 60% of the total SMA copolymers in the system. Over a short course of time, the bulge extracted 82 lipid molecules from the bilayer plane (Figure S11A) and became discshaped with the size of ca. 8 x 7 x 4 nm (Figure 5D). The protrusion remained stable until the end of the simulation, which implies that a single layer of SMA copolymers was sufficient to


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stabilize the SMALP rim. This simulation was also extended by 1 μs with polarizable water resulting in formation of even larger disc-shaped protrusion that almost separated from the bilayer at the end of the simulation (Figure S12). Within the observed SMALP-like disc, the SMA copolymers were arranged into two belts surrounding lipids as indicated by two peaks on the density plots for styrene and MA (Figure S13). At the same time, the inter-peak distances evidence that styrene groups interacted with the lipid core whereas maleic acid groups were facing the solvent, as suggested earlier9. For the sake of reproducibility, we have run a replica simulation yielding results, which were qualitatively similar to those described above, see e.g. Fig. S11A. The observed in the replicate simulation SMALP-like protrusion consisted of 96 lipids and 524 monomers (i.e., 84% of the total SMA copolymers, both values averaged over the last microsecond). Interaction of statistical 3:1 SMA copolymers with DMPC bilayers. Since real-life SMA copolymers have statistical sequences, we have also investigated the interaction of SMA copolymers of different sequences with the same bilayer. Sequences of all statistical copolymers were generated according to the penultimate model (as described in Methods) and contained styrene blocks with the length ranging from 2 monomers up to 10 monomers (relative abundances of different blocks are shown in Fig. S5). The length of each copolymer was 52 monomers and it equaled that of periodic 3:1 SMA copolymers described in the previous section. The simulation setup also matched one used for the simulations of periodic copolymers with bilayer. The simulation was performed in double replicate leading to the qualitatively similar results (Fig. S11B). Overall, in these simulations (no. 8, see Tables 1, S1) we observed similar results as in the case of periodic 3:1 SMA copolymers (Fig. 6A-C). An aggregate of SMA copolymers formed after ~100 ns of simulation time, adsorbed onto the bilayer and started to pull lipids from the membrane plane (from ~600 ns), which eventually led to formation of stable disc-shaped bulge in the both MD runs. The average number of lipids in the formed protrusion was 103 (106 in the replicate simulation, averaged over the last microsecond, Fig. S11B) and the amount of stabilizing SMA copolymers equaled 421 monomers (510 for the replica, averaged over the last microsecond), i.e. corresponded to 67% and 82% of the total SMA copolymers, respectively.


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Interaction of length-dispersed 3:1 SMA copolymers with DMPC bilayers. In addition to sequence variance, real SMA copolymers also feature high degree of chain length dispersity. We investigated interactions of such length-dispersed copolymers by running simulations with a mixture of periodic 3:1 SMA copolymers varying in their lengths (no. 9 in Table 1). We used four types of copolymers in these simulations, consisting of 13, 26, 52 and 104 monomers (refer to Table S1 for the sequences). The simulation setup included 3 molecules of each type, which corresponded to the dispersity of 1.51. The rest of setup reproduced previous simulations. Similar to simulations with the periodic and statistical 3:1 SMA copolymers, in both performed replicate simulations, we observed formation of disc-shaped membrane protrusions stabilized by SMA copolymers (Fig. 6D-F, Fig. S11C). These protrusions encompassed 82/96 lipids (for two replicas, averaged over the last microsecond), whereas their rims were stabilized by 311/385 S/MA monomers (averaged over the last microsecond, corresponds to 53%/66% of the total monomers in the system). Interaction of short-chain 3:1 SMA copolymers with DMPC bilayers. In order to check whether short-chain periodic 3:1 SMA copolymers were still able to form SMALPs, we investigated the interaction of the same DMPC bilayer with 48 periodic small [SSSM]3 copolymers with molecular weight of ~1.3 kDa. The total mass of 48 such SMA copolymers corresponded to that of 12 longer periodic 3:1 SMA copolymers used in simulation no. 6. In this case we also observed initial fast aggregation of copolymers followed by their adsorption at the DMPC bilayer (Fig. 6G). Upon the formation of contacts between the hydrophobic tails of lipids and SMA copolymer aggregates the latter started to disaggregate. In the course of this process, several lipids transiently formed a small protrusion (Fig. 6H), which disappeared after ~1 μs of the simulation time, apparently due to inability of short chain SMA copolymers to promote its further growth. At the end of simulation, all SMA copolymers became completely disaggregated and distributed evenly in the membrane (Fig. 6I). In contrast to simulations with the periodic 2:1 SMA copolymers of larger molecular weight (vide supra), we did not observe formation of transmembrane pores.


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Figure 3. Interaction of periodic SMA copolymers (2:1 ratio of styrene:MA residues, [SSM]17) with a DMPC bilayer. A: Initial configuration of the system; B: a snapshot upon the absorption of the SMA aggregates to the bilayer; C: the final configuration with a pore formed; D: growth of a membrane pore after 1 μs-long simulation with the polarizable water model, which extended the preceding 5 μs-long simulation with the standard water model. Water and ion beads are omitted for clarity. E: Enlarged view of porous defect of the DMPC bilayer stabilized by SMA copolymers. Water is shown as the blue surface. Hereinafter, phosphate, choline and MA moieties are shown as orange, blue and orange spheres, respectively, styrene rings – as yellow triangles, SMA copolymer backbone and lipids – as white and salmon sticks.


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Figure 4. Interaction of SMA copolymers (3:1 ratio of styrene:MA residues, [SSSM]13) with a DMPC bilayer. A: Initial configuration of the system; B: a snapshot upon the absorption of the SMA aggregates to the bilayer; C: the final configuration with a disc-shaped protrusion formed. Water and ion beads are omitted for clarity.


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Figure 5. Formation of the disc-shaped protrusion stabilized by periodic SMA copolymers (3:1 ratio of styrene:MA residues, [SSSM]13). A-C: Early steps of emergence of the discshaped protrusion. Contacts of the lipid tails with the hydrophobic styrene moieties of a SMA copolymer cluster are shown enlarged in the inset. D: Shape and dimensions of the disc-shaped protrusion at the end of the simulation. Water and ion beads are omitted for clarity.


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Figure 6. Interaction of statistical (A-C), length-dispersed (D-F) and short-chain ([SSSM]3) (G-I) 3:1 SMA copolymers with a DMPC bilayer. A, D, G: Snapshots upon the absorption of the SMA copolymer aggregates to the bilayer. B, E, H: Early steps of emergence of the discshaped protrusion. C, F, I: Final configurations.

Formation of SMALPs depends on size and sequence of SMA copolymers Here we have used coarse-grained MD simulations to investigate the molecular details of solution behaviour of SMA copolymers with 2:1 and 3:1 molar styrene:MA ratios and the interaction of these copolymers with model bilayers. In our simulations, we have observed all the steps which were previously proposed23 for the process of SMALP formation: (1) aggregation of SMA copolymers in a water environment; (2) their interaction with lipid membrane, adsorption on it and insertion into the bilayer, (3) solubilization of the membrane or formation of a SMALPlike disc. Importantly, we have demonstrated that the mechanisms by which SMA copolymers interact with membranes could be different depending on their composition.


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Our MD simulations of SMA copolymers in solution indicate that they form aggregates of limited size, which become heterogeneous at higher SMA concentrations (10 and 20 mM). At the same time, both the increase of copolymer unit charge and the increase of copolymer concentration led to destabilization of SMA copolymer clusters apparently due to accumulation of negative charges inside the SMA copolymer clusters. This excess negative charge cannot be compensated by counterions starting from a certain size of SMA copolymer clusters, which does not seem to exceed several nanometers according to results of our simulations. However, the mechanism of destabilization differs for various species of SMA copolymers. For the 3:1-2 SMA copolymers and singly charged 3:1 SMA copolymers at higher concentrations, individual copolymers became more stretched, while for 2:1 SMA copolymers, individual copolymers did not unfold, but the whole cluster swelled due to electrostatic repulsion of SMA copolymers. The observed tendency of SMA copolymers to form clusters of a finite size in solution might account for the narrow distribution of SMALP sizes, which was established experimentally in a number of studies9. If SMA copolymers exist, in solution, as small clusters containing up to several tens of typical molecules, such clusters, upon their interaction with the membrane, would only be able to extract membrane fragments of a particular size; this size would be limited by the number of copolymers in the cluster. In our modeling, 2:1 and 3:1 SMA copolymers demonstrated qualitatively different behavior. The interactions of 2:1 SMA copolymers with lipid membrane were akin to the initial steps of membrane solubilization by classical detergents53. Solubilization by detergents results in formation of micelles; we, instead, observed formation of pores, as in MD simulations of Xue and colleagues who also modeled the interaction of 2:1 SMA copolymers with membranes37. Although the similarity with results of Xue et al. justifies our modeling routine, no SMALP-like particles were seen to form in the MD simulations of 2:1 SMA copolymers. In contrast, 3:1 SMA copolymers (both periodic and statistical) induced formation of SMALPs according to a so far unobserved mechanism (Fig. 4-6). The SMA copolymers initially bound to the lipid membrane and then sequestered hydrophobic lipid moieties out of it; these sequestered moieties resembled small SMALPs with the diameter of ca. 8 nm. The process was driven


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mainly by hydrophobic interactions, as suggested earlier for formation of SMALPs23. Similar to the mechanism of translocation of amphiphilic nanoparticles across the membrane54,55, the process started at defects of lipid packing. From the first glance, the difference in the molecular mechanisms of interaction with membrane, as demonstrated here for 2:1 and 3:1 SMA periodic copolymers, contradicts numerous experimental observations on almost equal competence of 2:1 and 3:1 SMA copolymers in production of SMALPs22,56. We believe that this conundrum is only apparent and could be solved by a detailed comparison of experimental data and our findings as outlined in the following. In a thorough study of the dependence of the SMA copolymer efficiency on the styrene:MA ratio, Killian and colleagues studied four types of SMA copolymers, at different values of pH and ionic strength, by measuring the decrease in turbidity of a liposome solution in response to the addition of SMA copolymers46. In their study, the styrene to MA ratio varied from 1.4 to 4. At neutral pH, the 1.4:1 SMA copolymers were least efficient; they decreased the turbidity only by less than a half. 2:1 and 3:1 SMA copolymers made the solution completely transparent, whereas the 4:1 SMA copolymers were slightly less efficient.

The authors

concluded that 1.4:1 copolymers were too polar/charged (at neutral pH) to stabilize SMALPs, whereas SMA copolymers with larger styrene content could do that. Killian and colleagues also pointed out that the styrene to MA ratios reflect the "average fraction of the monomer units in the copolymer, but does not specify anything about the monomer sequence"46. In fact, real copolymers usually contain styrene blocks of various length, albeit in different proportions. Killian and colleagues estimated, for the four SMA copolymers they studied, the fractions of styrene blocks of different lengths46. Analysis of the respective diagram in Fig. 7 from their paper46 shows that the efficiency of SMA copolymers in solubilizing liposomes strikingly correlated with the relative fraction of three-styrene units in the copolymer (Table S2). Our MD simulations indicate that units with ≥ 3 sequential styrene monomers were involved both in maintaining the integrity of aggregates of SMA copolymers and in their interaction with hydrophobic constituents of the lipid bilayer (see e.g. Fig. 5B). These data imply that formation of SMALPs may require three- and, perhaps, larger (e.g., four-) styrene units as the main "drivers". Thus, in contrast to periodic 2:1 SMA copolymers, commercial random 2:1 SMA copolymers, as routinely used in experiments may generate SMALPs9,22,46, could efficiently


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extract lipid patches out of the membrane owing to the large fraction of units with ≥ 3 sequential styrene monomers present in them46. Our MD simulations, as well as the earlier simulations of Marrink and colleagues37, indicate that the interaction of periodic 2:1 SMA copolymer with the membrane is essentially a solubilization process similar to destabilization of bilayers by classical detergents. The latter process consists of four main stages, namely: (1) relatively rapid penetration of detergent monomers into the outer monolayer; (2) trans-membrane equilibration of detergent monomers between the two leaflets; (3) saturation of the bilayer by detergents and consequent permeabilization of the membrane; and, ultimately, (4) transition of the whole bilayer to thread-like mixed micelles53. For the 2:1 SMA copolymer we observed steps from (1) to (3); the solubilization, however, cannot be completed because SMA copolymers cannot substitute for detergents and form micelles. The size of the resulting particles of approx. 30 nm, which corresponds to the size of the smallest possible membrane vesicles (with diameters of 25-30 nm57) and membrane fragments, would be governed by physical forces that act on small fragment of lipid bilayer in the presence of SMA copolymers; the interplay of these forces deserves further studies. In contrast, SMA copolymers that contained units with ≥ 3 sequential styrene monomers could pull lipid patches out of the bilayer. This capacity was observed both with periodic 3:1 SMA copolymers of the same length (Fig. 4, 5, S11-S13) and with copolymers that were dispersed in the monomer sequence (Fig. 6A-C) and in length (Fig. 6D-F). Only very short 3:1 SMA copolymers disaggregated on the bilayer surface and did not form any stable SMALP-like protrusions. This observation indicates that hydrophobic interaction within copolymer aggregates and between the aggregates and the lipid membrane could be a kind of trade-off.

Upon interaction with the membrane, part of stabilizing styrene groups entered

alternative hydrophobic interactions with the lipid tails. If the number of units with more than two sequential styrene monomers per copolymer were too small, then the integrity of the aggregate could not be retained, as shown in Fig. 6F-G. In this case we also observed no poration of the lipid bilayer, which implied that the short SMA copolymers were unlikely to solubilize the bilayer (which agrees with the recent experimental results by Pardo et al.58.


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Hence, extraction of lipid patches was possible only if the SMA copolymer aggregates retained their integrity in spite of losing part of intrinsic, stabilizing hydrophobic interactions. Our data analyses indicate that a SMA copolymer should possess a certain threshold number of units with more than two sequential styrene monomers not to disaggregate upon binding to the membrane. Otherwise, aggregates could disaggregate, as it was observed when such styrene blocks were absent (Fig. 3) or when their number per copolymer was too small (Fig. 6E-G). Based on results of our MD simulations we would suggest the following answers to questions that were formulated in Introduction: (1) Formation of SMALPs proceeds in the following steps: (a) in solution, SMA copolymers cluster into aggregates of a finite size; (b) aggregates of SMA copolymers bind to the lipid membrane in a process that is driven mainly by hydrophobic interactions between styrene blocks and hydrophobic lipid moieties; (c) aggregates of SMA copolymers sequester lipid patches from the bilayer and form stable disc-shaped protrusions, which tend to further bud from the lipid bilayer. (2) In the observed disc-shaped membrane protrusions, interacting SMA copolymers (consisting together of 421±75 monomers, which comprised 69±11% of all SMA copolymers present in the corresponding system) were organized into two belts that embraced lipid patches containing 91±12 DMPC lipids (all values averaged over 6 simulations, i.e. no. 6, 8 and 9, each in two replicas, with the standard deviation provided). (3) At the same time, no major difference in the SMALP-forming mechanism was observed for copolymers that possessed, on average, sufficient number of units of ≥ 3 sequential styrene monomers. When this number, roughly, was more than ten, the mechanism of SMALP formation looked similar regardless of their monomer sequence and chain length.

Conclusions In the present study, based on using the MARTINI force field, we have developed coarse-grained models for SMA copolymers with 2:1 and 3:1 molar ratios of styrene to MA. These models were further used to investigate the solution behavior of SMA copolymers and to assess how their propensity to aggregate depends on their charge and concentration. Simulations of interactions of 2:1 (periodic) and 3:1 (periodic and statistical) SMA copolymers with model bilayers revealed


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qualitatively different mechanisms of membrane destabilization. While the 2:1 species induced formation of hydrophilic pores in the bilayer resembling early stages of membrane solubilization by classical detergents, both the periodic and the statistical 3:1 SMA copolymers were capable of extracting lipid patches from the membrane leading to formation of SMALP-like discs. The morphology of these discs, with two belts of SMA copolymers that stabilized the rim of the lipid patch, was similar to the arrangements proposed earlier in literature. Furthermore, the careful analysis of available experimental data allowed us to hypothesize that the efficiency of formation of SMALPs depends on the fraction of styrene units with ≥ 3 sequential styrene monomers in SMA copolymers used for their preparation, so that these styrene blocks serve as main “drivers” of lipid extraction from the membrane and subsequent formation of SMALPs. Altogether, our results show how the delicate balance of hydrophobic and hydrophilic units in SMA copolymers can affect their interactions with membranes. These observations are worth future theoretical and experimental studies and could help in rational design of SMA-based nanocarriers and agents for protein solubilization.

Acknowledgments The work was supported by the Ostpartnerschaftenprogramm of DAAD and RFBR grant no. 18504-12045 to P.S.O., M.E.B. and K.V.S. and by the DFG grant no. STE640/15 to H.J.S.

Supporting information 1. SI.pdf: supplementary figures S1-S13, supplementary tables S1-S2.

References (1) von Heijne, G. The Membrane Protein Universe: What’s out There and Why Bother? J. Intern. Med. 2007, 261 (6), 543–557. (2) Hammond, C. Cellular and Molecular Neurophysiology; Academic Press, 2014. (3) Lee, A. G. Lipid–protein Interactions in Biological Membranes: A Structural Perspective. Biochimica et Biophysica Acta (BBA) - Biomembranes 2003, 1612 (1), 1–40. (4) Tribet, C.; Audebert, R.; Popot, J. L. Amphipols: Polymers That Keep Membrane Proteins Soluble in Aqueous Solutions. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (26), 15047–15050. (5) Civjan, N. R.; Bayburt, T. H.; Schuler, M. A.; Sligar, S. G. Direct Solubilization of Heterologously Expressed Membrane Proteins by Incorporation into Nanoscale Lipid Bilayers. Biotechniques 2003, 35 (3), 556–560, 562–563.


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(6) Knowles, T. J.; Finka, R.; Smith, C.; Lin, Y.-P.; Dafforn, T.; Overduin, M. Membrane Proteins Solubilized Intact in Lipid Containing Nanoparticles Bounded by Styrene Maleic Acid Copolymer. J. Am. Chem. Soc. 2009, 131 (22), 7484–7485. (7) Orwick, M. C.; Judge, P. J.; Procek, J.; Lindholm, L.; Graziadei, A.; Engel, A.; Gröbner, G.; Watts, A. Detergent-Free Formation and Physicochemical Characterization of Nanosized Lipid-Polymer Complexes: Lipodisq. Angew. Chem. Int. Ed. 2012, 51 (19), 4653–4657. (8) Tonge, S. R.; Tighe, B. J. Responsive Hydrophobically Associating Polymers: A Review of Structure and Properties. Adv. Drug Deliv. Rev. 2001, 53 (1), 109–122. (9) Dörr, J. M.; Scheidelaar, S.; Koorengevel, M. C.; Dominguez, J. J.; Schäfer, M.; van Walree, C. A.; Antoinette Killian, J. The Styrene–maleic Acid Copolymer: A Versatile Tool in Membrane Research. Eur. Biophys. J. 2015, 45 (1), 3–21. (10) Bersch, B.; Dörr, J. M.; Hessel, A.; Antoinette Killian, J.; Schanda, P. Proton-Detected Solid-State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs. Angew. Chem. Int. Ed. 2017, 56 (9), 2508–2512. (11) Broecker, J.; Eger, B. T.; Ernst, O. P. Crystallogenesis of Membrane Proteins Mediated by PolymerBounded Lipid Nanodiscs. Structure 2017, 25 (2), 384–392. (12) Parmar, M.; Rawson, S.; Scarff, C. A.; Goldman, A.; Dafforn, T. R.; Muench, S. P.; Postis, V. L. G. Using a SMALP Platform to Determine a Sub-Nm Single Particle Cryo-EM Membrane Protein Structure. Biochim. Biophys. Acta Biomembr. 2018, 1860 (2), 378–383. (13) Orwick-Rydmark, M.; Lovett, J. E.; Graziadei, A.; Lindholm, L.; Hicks, M. R.; Watts, A. DetergentFree Incorporation of a Seven-Transmembrane Receptor Protein into Nanosized Bilayer Lipodisq Particles for Functional and Biophysical Studies. Nano Lett. 2012, 12 (9), 4687–4692. (14) Dörr, J. M.; Koorengevel, M. C.; Schäfer, M.; Prokofyev, A. V.; Scheidelaar, S.; van der Cruijsen, E. A. W.; Dafforn, T. R.; Baldus, M.; Antoinette Killian, J. Detergent-Free Isolation, Characterization, and Functional Reconstitution of a Tetrameric K Channel: The Power of Native Nanodiscs. Proceedings of the National Academy of Sciences 2014, 111 (52), 18607–18612. (15) Gulati, S.; Jamshad, M.; Knowles, T. J.; Morrison, K. A.; Downing, R.; Cant, N.; Collins, R.; Koenderink, J. B.; Ford, R. C.; Overduin, M.; et al. Detergent-Free Purification of ABC (ATPBinding-Cassette) Transporters. Biochem. J 2014, 461 (2), 269–278. (16) Logez, C.; Damian, M.; Legros, C.; Dupré, C.; Guéry, M.; Mary, S.; Wagner, R.; M’Kadmi, C.; Nosjean, O.; Fould, B.; et al. Detergent-Free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles. Biochemistry 2016, 55 (1), 38–48. (17) Swainsbury, D. J. K.; Scheidelaar, S.; Foster, N.; van Grondelle, R.; Killian, J. A.; Jones, M. R. The Effectiveness of Styrene-Maleic Acid (SMA) Copolymers for Solubilisation of Integral Membrane Proteins from SMA-Accessible and SMA-Resistant Membranes. Biochim. Biophys. Acta Biomembr. 2017, 1859 (10), 2133–2143. (18) Cuevas Arenas, R.; Klingler, J.; Vargas, C.; Keller, S. Influence of Lipid Bilayer Properties on Nanodisc Formation Mediated by Styrene/maleic Acid Copolymers. Nanoscale 2016, 8 (32), 15016– 15026. (19) Dominguez Pardo, J. J.; Dörr, J. M.; Iyer, A.; Cox, R. C.; Scheidelaar, S.; Koorengevel, M. C.; Subramaniam, V.; Killian, J. A. Solubilization of Lipids and Lipid Phases by the Styrene-Maleic Acid Copolymer. Eur. Biophys. J. 2017, 46 (1), 91–101. (20) Postis, V.; Rawson, S.; Mitchell, J. K.; Lee, S. C.; Parslow, R. A.; Dafforn, T. R.; Baldwin, S. A.; Muench, S. P. The Use of SMALPs as a Novel Membrane Protein Scaffold for Structure Study by Negative Stain Electron Microscopy. Biochim. Biophys. Acta 2015, 1848 (2), 496–501. (21) Lee, S. C.; Knowles, T. J.; Postis, V. L. G.; Jamshad, M.; Parslow, R. A.; Lin, Y.-P.; Goldman, A.; Sridhar, P.; Overduin, M.; Muench, S. P.; et al. A Method for Detergent-Free Isolation of Membrane Proteins in Their Local Lipid Environment. Nat. Protoc. 2016, 11 (7), 1149–1162. (22) Lee, S. C.; Khalid, S.; Pollock, N. L.; Knowles, T. J.; Edler, K.; Rothnie, A. J.; R T Thomas, O.; Dafforn, T. R. Encapsulated Membrane Proteins: A Simplified System for Molecular Simulation. Biochim. Biophys. Acta 2016, 1858 (10), 2549–2557.


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(23) Scheidelaar, S.; Koorengevel, M. C.; Pardo, J. D.; Meeldijk, J. D.; Breukink, E.; Killian, J. A. Molecular Model for the Solubilization of Membranes into Nanodisks by Styrene Maleic Acid Copolymers. Biophys. J. 2015, 108 (2), 279–290. (24) Hazell, G.; Gee, A. P.; Arnold, T.; Edler, K. J.; Lewis, S. E. Langmuir Monolayers Composed of Single and Double Tail Sulfobetaine Lipids. J. Colloid Interface Sci. 2016, 474, 190–198. (25) Swainsbury, D. J. K.; Scheidelaar, S.; van Grondelle, R.; Antoinette Killian, J.; Jones, M. R. Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability. Angew. Chem. Int. Ed Engl. 2014, 126 (44), 11997–12001. (26) Rothnie, A. J. Detergent-Free Membrane Protein Purification. In Methods in Molecular Biology; 2016; pp 261–267. (27) Long, A. R.; O’Brien, C. C.; Malhotra, K.; Schwall, C. T.; Albert, A. D.; Watts, A.; Alder, N. N. A Detergent-Free Strategy for the Reconstitution of Active Enzyme Complexes from Native Biological Membranes into Nanoscale Discs. BMC Biotechnol. 2013, 13 (1), 41. (28) Lindhoud, S.; Carvalho, V.; Pronk, J. W.; Aubin-Tam, M.-E. SMA-SH: Modified Styrene–Maleic Acid Copolymer for Functionalization of Lipid Nanodiscs. Biomacromolecules 2016, 17 (4), 1516– 1522. (29) Zhang, R.; Sahu, I. D.; Bali, A. P.; Dabney-Smith, C.; Lorigan, G. A. Characterization of the Structure of Lipodisq Nanoparticles in the Presence of KCNE1 by Dynamic Light Scattering and Transmission Electron Microscopy. Chem. Phys. Lipids 2017, 203, 19–23. (30) Rehan, S.; Paavilainen, V. O.; Jaakola, V.-P. Functional Reconstitution of Human Equilibrative Nucleoside Transporter-1 into Styrene Maleic Acid Co-Polymer Lipid Particles. Biochim. Biophys. Acta 2017, 1859 (5), 1059–1065. (31) Jamshad, M.; Grimard, V.; Idini, I.; Knowles, T. J.; Dowle, M. R.; Schofield, N.; Sridhar, P.; Lin, Y.; Finka, R.; Wheatley, M.; et al. Structural Analysis of a Nanoparticle Containing a Lipid Bilayer Used for Detergent-Free Extraction of Membrane Proteins. Nano Res. 2014, 8 (3), 774–789. (32) Zhang, R.; Sahu, I. D.; Liu, L.; Osatuke, A.; Comer, R. G.; Dabney-Smith, C.; Lorigan, G. A. Characterizing the Structure of Lipodisq Nanoparticles for Membrane Protein Spectroscopic Studies. Biochim. Biophys. Acta 2015, 1848 (1 Pt B), 329–333. (33) Marrink, S. J.; Jelger Risselada, H.; Yefimov, S.; Peter Tieleman, D.; de Vries, A. H. The MARTINI Force Field: Coarse Grained Model for Biomolecular Simulations. J. Phys. Chem. B 2007, 111 (27), 7812–7824. (34) Marrink, S. J.; Tieleman, D. P. Perspective on the Martini Model. Chem. Soc. Rev. 2013, 42 (16), 6801–6822. (35) Bochicchio, D.; Panizon, E.; Monticelli, L.; Rossi, G. Interaction of Hydrophobic Polymers with Model Lipid Bilayers. Sci. Rep. 2017, 7 (1), 6357. (36) Rossi, G.; Monticelli, L. Simulating the Interaction of Lipid Membranes with Polymer and LigandCoated Nanoparticles. Advances in Physics: X 2016, 1 (2), 276–296. (37) Xue, M.; Cheng, L.; Faustino, I.; Guo, W.; Marrink, S. J. Molecular Mechanism of Lipid Nanodisk Formation by Styrene-Maleic Acid Copolymers. Biophys. J. 2018, 115 (3), 494–502. (38) Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides †. J. Phys. Chem. B 2001, 105 (28), 6474–6487. (39) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79 (2), 926–935. (40) Bereau, T.; Kremer, K. Automated Parametrization of the Coarse-Grained Martini Force Field for Small Organic Molecules. J. Chem. Theory Comput. 2015, 11 (6), 2783–2791. (41) Orekhov, P. S.; Kholina, E. G.; Bozdaganyan, M. E.; Nesterenko, A. M.; Kovalenko, I. B.; Strakhovskaya, M. G. Molecular Mechanism of Uptake of Cationic Photoantimicrobial Phthalocyanine across Bacterial Membranes Revealed by Molecular Dynamics Simulations. J. Phys. Chem. B 2018, 122 (14), 3711–3722.


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(42) Wassenaar, T. A.; Ingólfsson, H. I.; Böckmann, R. A.; Tieleman, D. P.; Marrink, S. J. Computational Lipidomics with Insane: A Versatile Tool for Generating Custom Membranes for Molecular Simulations. J. Chem. Theory Comput. 2015, 11 (5), 2144–2155. (43) de Jong, D. H.; Baoukina, S.; Ingólfsson, H. I.; Marrink, S. J. Martini Straight: Boosting Performance Using a Shorter Cutoff and GPUs. Comput. Phys. Commun. 2016, 199, 1–7. (44) Morrison, K. A.; Akram, A.; Mathews, A.; Khan, Z. A.; Patel, J. H.; Zhou, C.; Hardy, D. J.; MooreKelly, C.; Patel, R.; Odiba, V.; et al. Membrane Protein Extraction and Purification Using StyreneMaleic Acid (SMA) Copolymer: Effect of Variations in Polymer Structure. Biochem. J 2016, 473 (23), 4349–4360. (45) Oluwole, A. O.; Klingler, J.; Danielczak, B.; Babalola, J. O.; Vargas, C.; Pabst, G.; Keller, S. Formation of Lipid-Bilayer Nanodiscs by Diisobutylene/Maleic Acid (DIBMA) Copolymer. Langmuir 2017, 33 (50), 14378–14388. (46) Scheidelaar, S.; Koorengevel, M. C.; van Walree, C. A.; Dominguez, J. J.; Dörr, J. M.; Killian, J. A. Effect of Polymer Composition and pH on Membrane Solubilization by Styrene-Maleic Acid Copolymers. Biophys. J. 2016, 111 (9), 1974–1986. (47) Buchak, B. E.; Ramey, K. C. Monomer Sequence Distribution in Styrene-Maleic Anhydride Copolymers. J. Polym. Sci. B Polym. Lett. Ed. 1976, 14 (7), 401–405. (48) Pionteck, J.; Reid, V.; Macknight, W. J. 10.1002/actp.1995.010460210. Acta Polym. April 1995, pp 156–162. https://doi.org/10.1002/actp.1995.010460210. (49) Klumperman, B. Mechanistic Considerations on Styrene--Maleic Anhydride Copolymerization Reactions. Polym. Chem. 2010, 1 (5), 558–562. (50) Klumperman, B.; O’Driscoll, K. F. Interpreting the Copolymerization of Styrene with Maleic Anhydride and with Methyl Methacrylate in Terms of the Bootstrap Model. Polymer 1993, 34 (5), 1032–1037. (51) Komarov, P. V.; Veselov, I. N.; Khalatur, P. G. Self-Organization of Amphiphilic Block Copolymers in the Presence of Water: A Mesoscale Simulation. Chem. Phys. Lett. 2014, 605-606, 22–27. (52) Yesylevskyy, S. O.; Schäfer, L. V.; Sengupta, D.; Marrink, S. J. Polarizable Water Model for the Coarse-Grained MARTINI Force Field. PLoS Comput. Biol. 2010, 6 (6), e1000810. (53) Lichtenberg, D.; Ahyayauch, H.; Alonso, A.; Goñi, F. M. Detergent Solubilization of Lipid Bilayers: A Balance of Driving Forces. Trends Biochem. Sci. 2013, 38 (2), 85–93. (54) Van Lehn, R. C.; Ricci, M.; Silva, P. H. J.; Andreozzi, P.; Reguera, J.; Voïtchovsky, K.; Stellacci, F.; Alexander-Katz, A. Lipid Tail Protrusions Mediate the Insertion of Nanoparticles into Model Cell Membranes. Nat. Commun. 2014, 5, 4482. (55) Bonhenry, D.; Dehez, F.; Tarek, M. Effects of Hydration on the Protonation State of a Lysine Analog Crossing a Phospholipid Bilayer - Insights from Molecular Dynamics and Free-Energy Calculations. Phys. Chem. Chem. Phys. 2018, 20 (14), 9101–9107. (56) Lee, S. C.; Pollock, N. L. Membrane Proteins: Is the Future Disc Shaped? Biochem. Soc. Trans. 2016, 44 (4), 1011–1018. (57) Feniouk, B. A.; Cherepanov, D. A.; Voskoboynikova, N. E.; Mulkidjanian, A. Y.; Junge, W. Chromatophore Vesicles of Rhodobacter Capsulatus Contain on Average One FOF1-ATP Synthase Each. Biophys. J. 2002, 82 (3), 1115–1122. (58) Domínguez Pardo, J. J.; Koorengevel, M. C.; Uwugiaren, N.; Weijers, J.; Kopf, A. H.; Jahn, H.; van Walree, C. A.; van Steenbergen, M. J.; Killian, J. A. Membrane Solubilization by Styrene-Maleic Acid Copolymers: Delineating the Role of Polymer Length. Biophys. J. 2018, 115 (1), 129–138.


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Maleic Acid Copolymers Form SMALPs by Pulling Lipid

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