Molecular Dynamics Simulations of the Initial Adsorption Stages of

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Molecular Dynamics Simulations of the initial Adsorption Stages of Fibrinogen on Mica and Graphite Surfaces Stephan Köhler, Friederike Schmid, and Giovanni Settanni Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03371 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 23, 2015

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Molecular Dynamics Simulations of the initial Adsorption Stages of Fibrinogen on Mica and Graphite Surfaces Stephan Köhler,†,‡ Friederike Schmid,† and Giovanni Settanni∗,†,¶

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Institut für Physik, Johannes Gutenberg-Universität Mainz, Graduate School Materials Science in Mainz, and Max Planck Graduate Center mit der Johannes Gutenberg-Universität E-mail: [email protected]

Phone: +49 6131-39-20492. Fax: +49 6131-39-20496

∗ To

whom correspondence should be addressed Gutenberg-Universität Mainz ‡ Graduate School Materials Science in Mainz ¶ Max Planck Graduate Center † Johannes

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Abstract

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Fibrinogen, a blood glycoprotein of vertebrates, plays an essential role in blood clotting by

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polymerizing into fibrin when activated. Upon adsorption on material surfaces, it also con-

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tributes to determine their biocompatibility and has been implicated in the onset of thrombosis

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and inflammation at medical implants. Here we present the first fully atomistic simulations of

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the initial stages of the adsorption process of fibrinogen on mica and graphite surfaces. The

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simulations reveal a weak adsorption on mica that allows frequent desorption and reorienta-

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tion events. This adsorption is driven by electrostatic interactions between the protein and the

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silicate surface as well as the counter ion layer. Preferred adsorption orientations for the glob-

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ular regions of the protein are identified. The adsorption on graphite is found to be stronger

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with fewer reorientation and desorption events, and showing the onset of denaturation of the

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protein.

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Introduction

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A major factor determining the biocompatibility of a material is the adsorption of proteins on its

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surface.

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quickly covered by a large variety of blood components in a competitive adsorption process. 2 In

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the case of nanoparticles, a protein corona is formed around them, whose composition depends

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on the nature of the particles and determines their functions. 3,4 More generally, reorientations or

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deformations of the adsorbed proteins, which expose or bury the protein’s functional epitopes or

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change their conformations, can affect the response of the organism to the introduced material. 5

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One of the most abundant adsorbing proteins is Fibrinogen (Fg). 2,6 Due to its involvement in

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the blood clotting cascade and immune response, its adsorption behavior crucially influences the

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biocompatibility of materials. 7–9

Materials in contact with blood, e.g. stents, medical implants or nanocarriers, 1 are

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Fg is a large (340 kDa) soluble fibrous glycoprotein present in the blood of vertebrates. After

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an injury, fibrinogen is activated by the enzyme thrombin and forms fibrin fibers. Fibrin fibers

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assemble into a mesh which is the basis of the blood clot. 10 Fg is a homo-dimer made of two 3 ACS Paragon Plus Environment

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protomers, each consisting of three non-identical peptide chains Aα, Bβ and γ that are covalently

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linked by disulfide bonds. The N-termini of the two protomers are covalently bonded one to each

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other by disulphide bridges and form the so called E-region. The N-termini of the Aα-chain

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and the Bβ chain contain longer flexible parts that are capped by the so-called Fibrinopeptides

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(Fp). Two symmetric three-stranded coiled-coil regions connect the E-region to the C-terminal

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ends of the protein. The central part of each coiled-coil region hosts a molecular hinge which

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conveys an extraordinary degree of flexibility to the protein. 11,12 The C-terminal region of the Aα-

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chain (αC-region) is mostly disordered and folds back toward the E-region. 13,14 Globular domains

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called β C-domain and γC-domain at the C-terminal end of the Bβ - and γ-chain, respectively, form

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the so called D region. The D region contains several integrin binding sites, including the P1

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and P2 sites (residues γ190-202 and γ377-395, respectively) which are known to bind leukocyte

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integrin αM β2 15,16 , and site H12 (residues γ392-411), which binds to the platelet integrin receptor

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αIIb β3 . 17 In particular, P1 is partly located in a cleft between the γC and β C domain (binding

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cleft). Additionally, the D region contains the a- and b-"holes" which are the binding sites of the

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"knobs" at the end of the Fp tethers of the E region and play a major role in fibrin formation. A

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schematic representation of Fg is shown in Figure 1(a).

Figure 1: (a) Schematic representation of Fg near a solid surface. The simulated part of the protein is colored in black. In (b) and (c) a close-up view of the D and E region, respectively, where the vectors used to characterize the orientation of regions with respect to the surface are indicated with red arrows. See main text for a detailed description.

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Several imaging techniques have been used to investigate fibrinogen adsorption. 18–27 In sev-


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eral of these experiments Fg shows a trinodular structure with a broad distribution of opening

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angles 18,24,26,28 highlighting the flexibility of the molecule. Two surfaces often used in these ex-

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periments are mica and graphite 18–24 . They represent an ideal charged/hydrophilic and a non-polar

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surface, respectively, and their sheet structure allows for the production of atomically flat surfaces.

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The techniques used for these experiments, however, do not allow to spatially resolve the mech-

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anism behind the flexibility of Fg or the atomic scale details of the adsorbed state. Simulations,

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which have been used to study protein adsorption on mica 29–31 and graphite/ graphene, 32–36 can

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help to fill the spatial resolution gap. The adsorption of the γC domain of Fg on various self assem-

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bled monolayer surfaces has been investigated using atomistic molecular dynamics (MD) simula-

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tions, which showed rolling motions but no deformations. 37 The adsorption of Fg on graphene has

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also been investigated using atomistic MD simulations, 36 which showed slow equilibration possi-

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bly driven by the formation of hydrophobic contacts and conformational rearrangements. Further

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simulations of Fg explored its mechanical response to external forces, 38,39 as well as its flexibil-

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ity in solution. 12 Fg adsorption has been also studied using simplified models, 40–42 where Fg is

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replaced by one or a small number of interacting objects that represent the whole molecule or the

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globular regions. Recently we have proposed a model of Fg adsorption, 12 which includes the flex-

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ibility observed in atomistic simulations. In this model the globular regions of Fg are represented

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as hard spheres and the coiled-coil regions as rods with one hinge on each side of the E-region.

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We have shown that this model can fit the experimentally observed distribution of opening angles

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(the angle formed by the three globular regions of Fg adsorbed on the surface) on mica, only by

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assuming the presence of a correlation between the two hinges on the two Fg protomers. The most

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probable reason for the emergence of these correlations is a preferred adsorption orientation of the

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Fg protomer for the charged mica surface, possibly driven by asymmetric charge distribution on

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the surface of the protein. 12

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Here we report on atomistic molecular dynamics simulations of the initial stages of Fg adsorp-

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tion on mica and graphite. In these simulations we address the speed, strength and reversibility of

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the adsorption process on both surfaces, as well as the emergence of preferential adsorption orien-


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tations for the Fg protomer. We also address the change in the flexibility of Fg upon adsorption as

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well as the possible onset of deformation/denaturation.

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Simulation Methods Table 1: Initial simulation box sizes, particle numbers and length of the trajectories for the different systems of fibrinogen. System Mica, 0 Mica, 120 Mica, 240 Graphite, 0 Graphite, 120 Graphite, 240

Initial box size [nm]

N Simulation time [ns] 681900 120, 120, 95, 94, 71, 70, 48, 48, 45, 43 18.47 × 29.91 × 14.30 681937 120, 96, 91, 90, 85, 70, 45, 45, 44, 43 681909 120, 100, 70, 70, 70, 46, 45, 41, 38, 30 440893 51,48, 45, 20, 20, 18 12.18× 27.36 × 13.03 440875 52, 50, 49, 49, 26, 19 440698 51, 49, 48, 44, 26, 23

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A high resolution crystal structure of human fibrinogen is available 43 and served as a starting

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point for our simulation. Only the atoms in the crystal structure were retained. Thus, the N-termini

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of all chains, as well as the αC region and the carbohydrate cluster attached to the coiled-coil

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region, were not included in the simulation. The crystal structure was terminated with neutral

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N- and C-termini for each chain. The Fg dimer was equilibrated in a surrounding water box at

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physiological NaCl concentration (0.15M) and simulated for 10ns as described previously 12 using

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the CHARMM27 force field with carbohydrate extensions 44–47 and NAMD. 48

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Mica and graphite were chosen as model solid surfaces to investigate the adsorption behavior

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of fibrinogen. The graphite surface was built as a 6 layer graphene sheet using the Carbon Nanos-

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tructure Builder within the program VMD. 49 The carbon atoms in graphite have been defined as

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uncharged, with a Van der Waals (VdW) radius of 0.19924 nm and VdW energy well of -0.07

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Kcal/mol. 44 Standard CHARMM combination rules apply for the VdW radii and VdW energy

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wells to determine the VdW potential between pairs of atoms. The other parameters such as bond,

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angle and dihedral parameters are given in Supplementary Information. The mica surface was

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constructed according to a recently published model 50 which was successfully adopted to simu-


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late peptide adsorption 51,52 . The mica surface consists of a two-layer sheet with realistic surface

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defects. The defects are point defects where an aluminum atom substitutes a silicon atom. The

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model assigns a positive partial charge of 1.0e to unbound potassium ions, positive partial charges

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of 1.1e, 0.8-1.45e (depending on connectivity and 0.2e to silicon, aluminum, and hydroxyl hydro-

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gen atoms, respectively, and negative partial charges ranging from -0.783e to -0.55e (depending on

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connectivity) to oxygen atoms. The other parameters of the force field are given in Supplementary

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Information. The simulation box for the surface system is periodic in all dimensions. The solid

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surface was constructed as being continuous in the xy-plane by defining covalent bonds that wrap

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around the periodic boundaries.

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The initial mica structure has a potassium counter ion layer on only one side of the solid

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surface. To achieve an even ion distribution, a slab of mica was simulated in vacuum under periodic

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boundary conditions. In a first step the system was simulated at 1350K with the electrostatic

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interaction turned off. This lead to a uniform distribution of the potassium ions in the vacuum. In a

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second step the electrostatic interactions were turned on and several iterations of alternating short

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MD simulations and energy minimizations were performed while reducing the temperature. This

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generated a mica slab with evenly distributed potassium ions on the two sides.

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A 12nm high water box with 0.15M NaCl was constructed on top of the solid surface using

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VMD. Water molecules were explicitly modeled using TIP3P. 53 The van der Waals forces were

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cut off at 1.2nm and PME was used for long range electrostatic interactions with a grid spacing

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of 0.1nm. The covalent bonds involving hydrogen atoms were fixed in length and a 2fs time

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step was used for the integration of the equations of motion. The energy of the surface system

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was minimized and an equilibration run of 0.6 ns was performed at constant pressure (1 atm)

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and temperature (310 K) with a Langevin thermostat and a Langevin piston barostat 54,55 using

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1 ps and 0.1 ps as decay time, respectively. The dimension of the box was allowed to fluctuate

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only in the z-direction to maintain constant pressure. The average electrostatic potential along

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the direction perpendicular to the slab was measured by solving the Poisson equation using the

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PMEPOT plugin 56 of VMD on the last 0.2 ns of the equilibration trajectory with a grid spacing of


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0.03 nm and an Ewald factor of 1.0. After the equilibration the first protomer and residues α27-

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65,β 58-95 and γ14-40 of the second protomer (see Figure 1) were added to the water simulation

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box. No C-termini patches were applied to the cut in the second protomer, which provides polar

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but uncharged termini. The protein was added in such a way that the minimal distance to the

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solid surface was at least 0.8nm. Three different initial orientations (labeled 0, 120, 240) were

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constructed by rotating Fg around its long axis by 120◦ . This procedure limits the sampled space

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to so called side-on adsorption which is known to be the dominant adsorption mode, at least in

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the dilute regime. 24,41 The orientation 0 is positioned in such a way that the carbohydrate cluster

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points away from the solid surface. For orientation 240 the carbohydrate cluster points toward the

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surface and are located between surface and coiled-coil region. The orientation 120 is intermediate

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and has the cleft in the D-region pointing away from the surface. Due to the orientation algorithm,

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the different orientations on mica and graphite are similar to each other but vary by a few degrees.

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The positions of the heavy atoms of the protein and the solid surface were fixed during solvent

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minimization. The constraints were gradually released over a 0.75ns equilibration period. After

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that, several isobaric, isothermal MD simulations were performed using NAMD (see Table 1).

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The longest simulations reach 120ns. This is still well below the timescales typically probed

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in experiments. 22,24 Our simulations can thus only give informations about the initial stages of

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fibrinogen adsorption. A harmonic potential was applied to the protein atoms along the z direction

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when they moved higher than 10nm above the solid surface to prevent adsorption on the periodic

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image of the surface. This force was implemented using the tclForces module of NAMD. To

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mimic the effect of bulk material, the lower sheet in mica and the lowest two sheets in graphite

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were restrained to their initial positions using a 3kcal/mol harmonic restraint. The water density

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profile along the z direction measured on a 2nm×2nm-wide slab for 0.5 ns showed no difference

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at the two sides of the slab. Hence the interplay between the pressure coupling algorithm and the

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harmonic constraints on the slab did not cause noticeable artefacts.

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In the case of graphite, the force field and simulation setup described above leads to trajectories

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which show good agreement with recent simulations by Chong et al of the adsorption of fibrinogen


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on graphene 36 regarding the kinetics of the adsorption process as monitored by the number of

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contacts and the amount of buried protein surface upon adsorption (see Figure S1 in Supplementary

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Information), and the emergence of π-π interactions (see below in the Results and Discussion

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Section). In the case of mica, the conditions used in the present study match more closely those

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used in Ref. 30 (section IV), i.e. the case where counter ions were added to the simulation box

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to neutralize the overall charge of the system (a role played by the potassium ions in the present

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case). The simulations of Trp-cage in that study showed desorption events and no unfolding upon

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adsorption, similar to what is observed in the present study for fibrinogen. The results of these

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comparisons provide a quality control for the present simulations.

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The graphite simulations were run for up to 50ns because this time length is sufficient to ob-

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serve large contact formation between protein and solid surface. The trajectories of the adsorption

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on mica were extended up to 120ns to improve the sampling of the adsorption/desorption events

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which do not occur on graphite on these time scales. In a few simulation runs, periodic images of

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the protein started to interact with each other. This can happen due to the rotational diffusion of the

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protein in the elongated periodic simulation box. To avoid artefacts, the corresponding trajectories

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were truncated just before the periodic interactions set in, i.e. distance between any pair of atoms

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from each protein image lower than 1.2 nm.

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The motion of Fg was studied using Principal Component Analysis 57 and the overlap of the

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essential dynamics subspace was used to quantify the similarity of the dominant motions across

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the different systems/simulations. 58 The hinge bending was characterized by a bending angle γ

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and dihedral angles ϕ as defined previously 12 (see Figure 2). The three groups of atoms used to

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define the γ angle are the E region (α50–58,β 82–90,γ23–31), the hinge region (α99–110, β 130–

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155,γ70–100), and the D region (β 200–458,γ140–394). The four groups of atoms used to define

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the ϕ angle are one part of the E region (α50–58,γ23–31), another part of the E region (β 82–

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90,γ23–31), the hinge region (α99–110, β 130–155,γ70–100) and the D region (β 200–458,γ140–

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394)

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The flexibility provided by the hinge allows the D and E regions to reorient more or less in-


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dependently with respect to the solid surface. For this reason their orientations are investigated

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separately. To characterize the different adsorption orientations we defined an angle θ describing

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the tilting of a relevant axis of the region with respect to the surface and an angle φ describing the

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rotation around the identified axis (Figure 1(b,c)). Both θ and φ are defined separately for the D

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and E regions. In the D region the main axis (xa ) connects the geometrical center of the γC domain

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to the geometrical center of the β C domain. A second vector (xb ) is defined pointing from the

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attach point of the coiled-coil region (residues α162-168β 193-202,γ136-141) to the midpoint of

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the first vector. In the E-region the main axis xa is defined as the vector connecting the geometrical

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center of the group (α55-65,β 85-95,γ30-40) in the first protomer with the center of the same group

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in the second protomer. The second vector points from the geometrical center of residues γ14-21

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of both protomers to the midpoint of xa . Without loss of generality, a coordinate system can be

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chosen such that xa is in the xz-plane. The angle θ is then defined as the angle between xa and the

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surface (xy-plane). To calculate φ a vector ζ perpendicular to xa and the unit vector in y-direction

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is constructed. The rotation angle φ is calculated as the angle between ζ and the projection of xb

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into the ζ y-plane. Thus, φ describes the rotation of xb around xa .

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A contact between the protein and the solid surface is defined when a heavy protein atom comes

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closer than 0.5 nm to the surface, or more precisely, when the z coordinate of the heavy protein

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atom is less than 0.5 nm above the average z-coordinate of the top layer of the solid surface. If

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such a contact is formed in the globular D and E regions and persists longer than 1 ns, we call

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this an adsorbed state. A contact between a given residue and the solid surface is called persistent

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if it forms in all sets of simulations, independent of the starting configuration.

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for an unbiased adsorption process, we also measured the fraction of heavy atoms on the protein

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surface contributed by each residue type: charged residues contributed 52% of the surface atoms,

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polar uncharged 33%, hydrophobic 11.5% and the carbohydrate groups 3.5%. Here protein atoms

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were considered as being on the protein surface if they were within 0.2nm of water atoms. To

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detect spreading of the globular regions of the protein during an adsorption event, we monitored

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the ”domain height”, which we define as the z-component of the distance between the center of


As a reference

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mass and that protein atom which is closest to the surface of the material.

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Results and Discussion

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Mica

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The dominant large scale motions of Fg on the mica surface (as identified using PCA), are bend-

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ing motions at the hinge, which closely resemble those previously described for Fg in solution. 12

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A large essential dynamics (ED) overlap (0.69–0.71) is observed between the three largest PCA

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modes measured in the different sets of simulations. The overlap with previously reported solu-

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tion simulations is also high (0.63). Furthermore the sampled distribution of hinge conformations

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shown in Figure 2(a) is in reasonable agreement with the corresponding distribution in solution, if

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one takes into account the limited sampling in both cases. The observed bending time of (16 ± 6)ns

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is also in agreement with the result in solution We thus conclude that the hinge motion close to

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a mica surface is very similar to that in solution, which supports one of the central assumptions

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adopted in our simplified model for Fg adsorption on mica. 12

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Although the hinge bending is similar in solution and at a mica surface, it can be the initial

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cause of contact formation between Fg and the surface. In addition, even if the protein has already

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formed contacts with the surface, the hinge bending can provide further mobility to the globular

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regions. In some instances this can lead to sliding and rolling motions on the surface (See Movie

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S1 in the supporting information). In a previous simulation of the γC domain 37 of Fg a rolling

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motion has been observed. To our knowledge the sliding motion has not been observed previously

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on this system.

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In simulations on mica, the total number of contacts formed with the surface reaches a plateau

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at ≈ 15 contacts (average over the simulations) after about 30ns (Figure 3(a)). The fraction of

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contacts formed by the different types of residues resembles essentially the one expected from

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the surface distribution of residues – the only exception being that positively charged residues

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contribute more than would be expected while the contribution of negatively charged and polar 11 ACS Paragon Plus Environment

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(a)

(b)

Figure 2: (a) Distribution of the hinge bending and dihedral angle at a mica surface. The definition of the angles is shown in the inset. (b) Maximally bent state of Fg at the mica surface (highlighted in (a)). A collision of the D and E regions prevents further bending at the hinge. The Aα, Bβ and γ chains from the whole protomer are rendered in blue, red and green, respectively, the carbohydrates in orange. For clarity the solvent is not shown. 1

residues is slightly lower than expected. This phenomenon is explained by the negatively charged

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nature of the mica surface. Since the exposed hydrophobic residues cover only a small fraction

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(11%) of the total surface of the protein and are, to a first approximation, evenly distributed over

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it, the free energy penalty to have them facing the charged mica surface is possibly not sufficient

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to cause a reorientation of the protein. Therefore, they are not under-represented in the contact

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counts.

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and reorientation events. On average a globular region was only adsorbed for (14 ± 3)ns before

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leaving the surface again. We observed a total of 51 adsorption events in the D region and 45 in

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the E region. The great flexibility provided by the hinge allow us to treat these events separately.

Another significant feature of the simulations is the observation of frequent desorption

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Similarly, the adsorption orientation of the D- and E-regions can be analyzed separately. Sev-

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eral different adsorption orientations have been identified for both globular regions by dividing the

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space of the adsorption angles into a small number of adsorption orientation states (Figure 4(a-b)). 12 ACS Paragon Plus Environment

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Figure 3: Average total number of contacts and fraction contributed by the different types of residues during adsorption on (a) mica and (b) graphite. The straight horizontal darkened lines represent the expected fractions according to the exposed surface area in the crystal structure (after equilibration). 1

For all simulated systems, the adsorption orientation states of the D- and E-regions overlap signifi-

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cantly (Table 2), although a bias towards the initial orientation state is certainly visible. These data

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clearly indicate that transitions from one orientation state to another occur frequently. We observed

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68 reorientation events (changes in the adsorption orientation state) for the D-region and 22 for the

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E-region. Neglecting trajectories where the D- (E-) region never contacted the surface this gives

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an average time of 27ns (14ns) between reorientation events.

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More specifically, the E region shows three distinct adsorption orientation states. The orien-

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tation E1 is significantly populated in all sets of simulations. In cases where the simulation starts

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with this orientation it never leaves it, while simulations starting with the other orientations often


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Figure 4: (a),(b) Distribution of the adsorption angles for the D- and E-region, respectively, and schematic representation of the corresponding adsorption orientation states. Chains are color coded as in Figure 2. The orange patch between the β C and γC domain indicates the binding cleft while the purple region identifies the P2 and H12 binding sites. In orientation D4 the P2 and H12 binding sites face away from the surface and are available for binding. (c) The residues α27-28,α38,α92,β 345-348, β 361-363, β 365, γ323, γ356-357), γ361 and carbohydrates 479-480 from the first whole protomer and α27-30, α37-38, α63-65, β 91 and γ38-40 from the second truncated protomer form persistent contacts on mica regardless of initial orientation (red licorice). The carbohydrate cluster (glycans) attached to the β C domain is shown in grey licorice, the P1 site is rendered in orange and the P2 and H12 sites in purple.(d) Example snapshot of a pair of oppositely charged amino acids (lysine in blue, aspartic acid in green) anchoring the E region in an Fp-down orientation. The aspartic acid interacts with a sodium ion (gray) that has replaced potassium (pink) from the counter ion layer. Hydrogen bonds between the lysine and the silicate ring are indicated by the black dotted lines. For clarity only the topmost atoms of the mica surface are shown.


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Table 2: Fraction of the time spent in each of the adsorption orientation states defined in Figure 4(a),(b). States containing the initial orientation are in bold. System D1 D2 D3 D4 E1 E2 E3 Mica, 0 33% 28% 5% 34% 46% 44% 10% Mica, 120 0% 60% 37% 3% 13% 28% 59% Mica, 240 8% 0% 42% 50% 100% 0% 0% All 16% 32% 25% 27% 56% 21% 23%

1

show reorientation towards E1. These data support the idea that E1 is a preferred adsorption orien-

2

tation. In the E1 orientation the flexible Fp tethers point toward the surface. The presence of many

3

charged residues in this region likely explains the preference for this adsorption orientation. In

4

simulations starting from orientation E1 (Mica/240), 60% of the Fg-surface contacts are provided

5

by the E region. In Mica/0 and Mica/120 simulations, these numbers are significantly lower (30%

6

and 19%), further supporting that E1 is indeed the preferred adsorption state.

7

The D-region shows several populated adsorption orientation states. In this case, a preference

8

for the orientation D4 is detectable. D4 is observed in all sets of simulations, although no set of

9

simulation starts from there. This orientation corresponds to an adsorbed state where the binding

10

cleft between γC- and β C-domain (containing part of the P1 site) is facing the surface, while the

11

sites P2 and H12 face the solution. This may have consequences on the accessibility of the integrin

12

binding sites upon adsorption on mica. Indeed, the preferential exposure of the P2 and H12 binding

13

sites may explain the integrin-mediated adhesion forces measured in recent AFM experiments of

14

leukocytes 59 and platelets 9,23 on fibrinogen-coated mica surfaces.

15

Further support for the presence of preferred adsorption orientations comes from the identifi-

16

cation of persistent contacts, i.e., contacts that are observed in all sets of simulations (Figure 4(c)).

17

The position of the residues involved in persistent contacts clearly highlights a preferred adsorption

18

orientation of the D region. All persistent contacts in the D region are located on the side expected

19

to contact the surface in orientation D4. Two persistent contacts are found in the carbohydrate clus-

20

ter. This cluster is mobile enough to form these contacts in the orientations D2–4. The persistent

21

contacts in the E region are mostly located in the Fp-tethers, thus corresponding to a E1 adsorption


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1

orientation. The truncated ends of the second protomer also form persistent contacts, which are

2

likely artefacts due to the truncation of the sequence. Interestingly, the persistent contacts in the

3

D region are not only formed by residues belonging to the large charged patches. 12,60 Instead, we

4

observe two more contact patches in the β C domain. The persistent contacts in the γC domain are

5

formed by one of the flexible loops of the a-hole. Most of the persistent contacts are formed in

6

regions where the side chains of positively and negatively charged residues are in close proximity,

7

(with the exception of the Asp-Glu-Asp motif of residue α37-39). Further investigation shows

8

that opposite-charge pairs provide an ideal balance for the negatively charged mica surface with its

9

positive counter ion layer. The negative side chain can trap ions from the counter ion layer, while

10

the positive side chain interacts directly with the surface. The interactions of lysine with mica

11

are particularly favorable as the positive charge of the amine is attracted to the negative charge

12

of silicate rings while it can also form hydrogen bonds with the oxygen atoms (see Figure 4(d)).

13

Visual inspection of the simulations shows that a single lysine contact can be enough to anchor the

14

E-region. The strong interaction of lysine with silica surfaces has already been reported in previous

15

simulations. 61 The same mechanism has also been observed during the adsorption of the Fg γC

16

domain on charged self assembled monolayers. 37

17

The fraction of contacts contributed by the D region is slightly smaller than what would be

18

expected from its surface area (34% observed, 41% expected). The discrepancy is more dramatic

19

for the charged patches. The negatively charged patch, corresponding to the a-hole involved in

20

fibrin formation, contributes 2.7% while 7.9% would be expected. The smaller positive patch on

21

the opposite side of the D region contributes 1.6% instead of the expected 4.2%. Notwithstanding,

22

the charged patches contribute some of the persistent contacts with the mica surface and they

23

may play a more subtle role in determining the adsorption orientation through their long range

24

Coulombic interactions with the surface. To verify this, the electrostatic potential and field of

25

the solvated mica surface was calculated (Figure 5(a)). At distances shorter than 1nm from the

26

surface, the electric field shows dumped oscillations due to the ion and water solvation layers and

27

becomes flat at larger distances. This complex electrostatic behavior creates a non trivial pattern of


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interactions with the protein which is further complicated by the non uniform charge distribution

2

on the mica surface (Figure 5(b)). Due to the fast decay of the electric field with the distance from

3

the surface, electrostatics play only a small role in the orientation of Fg away from the surface.

4

Indeed, the dipole distribution of the D region for desorbed Fg shows only a weak tendency to

5

point to the surface, with negative values of its z component dz (Figure 5(c-d)). On the other

6

hand, upon adsorption, the electric dipole of the D region tends to orient itself perpendicular to

7

the surface, either pointing to it (negative dz ), which corresponds to adsorption orientation D2,

8

or, to a larger extent, away from it (Figure 5(d)), which corresponds to adsorption orientation

9

D4. Thus, in the D4 adsorption orientation, the dipole of the D-region seems to align with the

10

mostly positive electric field above the surface generated by the layer of sodium and potassium

11

counterions. However, the field decays very quickly and may only affect the region of the protein

12

close to the surface. Given the interaction pattern involving pairs of oppositely charged residues

13

identified above, the preference for the D4 orientation could also be related to the number of such

14

pairs facing the surface in orientation D4, which is larger than on the opposite side (34 pairs versus

15

26).

16

Graphite

17

As for mica, the hinge behavior of Fg at a graphite surface is consistent with the behavior in the

18

solution simulations. The overlap between the PCA modes in the three orientations is slightly

19

lower than for mica (0.65 overlap). Comparing the behavior at graphite with the one observed in

20

the solution simulations, we find that the bending has similar overlap (0.65 overlap). The same

21

holds for the angle distribution shown in Figure 6(a) and the bending time of (17 ± 5)ns.

22

The adsorbed orientations can be grouped into the same classes as on mica. However, almost

23

no trajectory samples multiple orientations (see Figure 6(b),(c)). Only one trajectory shows evi-

24

dence of a reorientation event. The orientations of adsorbed molecules are mostly determined by

25

the starting orientations at the beginning of the simulations. All observed deviations are due to

26

reorientations occurring before adsorption. Thus, we cannot discuss desorption or reorientation 17 ACS Paragon Plus Environment

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Figure 5: Electrostatics at the mica surface. (a) Average component of the electrostatic field perpendicular to the mica surface as a function of the distance z from the surface (top box) and concentration of the ions and water molecules relative to their bulk concentration (lower boxes). (b) Electrostatic potential on the hydrated mica surface observed from top. The isosurfaces are shown at ±26.7mV (blue/red). (c) Average dipole moment (blue arrow) of the D region of Fg superimposed on its structure. The coloring scheme of the chains is the same as in Figure 2. (d) Distribution of the z component of the dipole of the D region for adsorbed and desorbed fibrinogen conformations (the total dipole moment of the D region is 15 ± 2 e·nm) 1

times in this case.

2

We have observed 13 adsorption events of the D region and 11 for the E region. In only

3

three of these cases was a desorption observed and in only one case did the desorption occur after 18 ACS Paragon Plus Environment

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Figure 6: (a) Distribution of the hinge bending and dihedral angle at a graphite surface. Characterization of adsorbed orientations on graphite: (b) Histogram of the orientation of the E region. (c) Histogram of the orientation of the D region. (d) Residues γ74, γ297, γ355-360 and carbohydrate 476 form persistent contacts on graphite regardless of the starting orientation (red licorice). The carbohydrate cluster is rendered as grey licorice, the P1 site in orange and the P2 and H12 sites in purple. 1

adsorption events lasting more than 1 ns. Very few reorientations were observed on graphite. This

2

supports the idea that graphite is "stickier" than mica for Fg. As discussed above, the hinge bending

3

can provide the impetus to bring the globular regions of Fg close to the surface. In the case of

4

graphite this leads to almost irreversible adsorption events. Thus a bending event not occurring in

5

a plane parallel to the surface will lead to irreversible contact formation and hinder further bending

6

motions, which eventually influences the sampling of the hinge angles and the bending modes.

7

The persistent contacts are limited to an isolated residue in the disordered part of the hinge

8

region, the carbohydrate group and the flexible loops of the a-hole ( Figure 6(d)). Since the a-hole

9

is located at the tip of the D-region, it can be brought into contact with the surface from several

10

initial orientations through hinge motions. The existence of persistent contacts in this region is

11

thus more likely a geometric effect than the result of specific interactions. It should be noted that

12

no persistent contacts are formed in the E-region.

13

The sticky nature of the graphite surface is also supported by the large number of contacts


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1

formed between Fg and the graphite surface (Figure 3(b)). In fact, the number of heavy atoms in

2

contact with the surface is almost an order of magnitude larger than in the case of mica. It is also

3

interesting to note that the distribution of contacts markedly differs from that on mica. As expected,

4

the fraction of hydrophobic contacts between the protein and the graphite surface is larger than their

5

fraction on the solvent exposed surface area. In turn, the contribution of the charged residues to

6

contacts with the graphite surface is reduced. In fact, the overall distribution of the contacting

7

residues is closer to the distribution of residues in the whole protein (41% polar, 26% charged,

8

33% hydrophobic), not just the protein surface. About half of the aromatic residues involved in

9

contacts with the surface form π − π interactions, in agreement with previous simulation data for

10

the adsorption of proteins on graphene. 35,36 It is worth noting that, given the short cutoff used for

11

the definition of the contacts (0.5nm) and the structure of the graphite hydration layer (Figure S2

12

in Supplementary Information), the formation of a contact occurs mostly by displacement of water

13

molecules from the first hydration layer of the graphite. (a)

(b)

(c)

Figure 7: (a) The adsorbed conformation of Fg on graphite shows a noticeable flattening of the domains. Coloring as in Figure 2(b). Histogram of the change in domain height for (b) mica and (c) graphite.

14

A pronounced flattening of the adsorbed domains is observed upon adsorption on graphite (Fig-

15

ure 7). This is likely the onset of denaturation, which is known to occur if Fg adsorbs to hydropho-

16

bic surfaces. 62 The denaturation of globular domains leads to their spreading on the surface. As an

17

indication of this we monitored the change in domain height (the domain height has been defined


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in the Methods section) during individual adsorption events. In mica, this difference is centered

2

around zero, indicating that domains do not spread (Figure 7(a)). On graphite, however, changes

3

are more pronounced and lead systematically to a reduction in domain height (Figure 7(b)). This

4

trend is in line with experimental findings according to which the domain height changes very little

5

if Fg is adsorbed on mica, but is reduced on graphite. 22 It should be mentioned that changes in the

6

domain height on mica can also occur as a result of rolling of the non spherical domains. The

7

electric field on the charged mica surface is unable to induce unfolding during the simulation time,

8

mostly because it is neutralized by the counterion layers. 30

9

A significant element missing in our simulations is the αC region, which was not resolved in the

10

crystal structure of Fg. 43 Recent AFM experiments comparing full length Fg with truncated Fg that

11

lacks the αC region proved that the αC region does not play a critical role during the adsorption of

12

the first Fg monolayer on mica 63 . It mostly influences the formation and the properties of multiple-

13

Fg layer matrices. Neither these experiments or our simulations, however, can exclude that the

14

presence of the αC region may alter the orientational adsorption of Fg or the degree of exposure of

15

its functional epitopes upon adsorption. The N-terminal parts of the Aα and Bβ chains, which are

16

also disordered and were not modelled in our simulations, may have favorable interactions with the

17

mica surface due to their hydrophilic nature. However, due to their flexible structure, it is unlikely

18

that they introduce an orientational bias in the adsorption process, although their presence may

19

weaken the bias observed in this work for the E region on mica. In the present simulations, the

20

carbohydrate cluster on the β C domain contributes persistent contacts to the mica surface, thanks

21

to its flexible nature. Assuming that the carbohydrate cluster in the coiled-coil region (not modelled

22

in the present simulations) has a similar behavior, this may provide a weak bias towards certain

23

adsorption orientations for the coiled-coil region. The impact on the adsorption orientation of the

24

D region will however be relatively minor, because of both the large flexibility of the carbohydrate

25

cluster and of the coiled-coil region in-between.

26

The length of the simulations reported in the present work, which is short compared to the

27

time measured experimentally, only allow us to address the initial adsorption stages. To mitigate


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1

this problem, we have accumulated large statistics by running several simulations starting from

2

different initial orientations. Although the single simulation will not necessarily show adsorption

3

along the most probable pathway, the statistics obtained from many simulations using significantly

4

different initial conditions will likely be biased towards the most probable routes, thus increasing

5

considerably the statistical significance of the the results exposed here.

6

Conclusion

7

We have used molecular dynamics simulations to investigate the initial adsorption stages of Fg on

8

mica and graphite surfaces. In both cases, the simulations reveal a hinge bending mechanism that

9

is consistent with the behavior observed in solution, thus confirming one of the central assumption

10

behind our previously published simplified model of Fg adsorption. 12 Our simulations also help to

11

identify the sources of an asymmetry in the orientation of Fg adsorbed on mica that was predicted

12

by our simplified model. The adsorption on mica is found to be dominated by electrostatic effects

13

generated by the charged surface and the first solvation layers composed of positive counterions

14

and water molecules. Indeed, pairs of positively and negatively charged residues are found to

15

drive the adsorption by establishing interactions with both the solid surface and the counter ion

16

layer. The preferred adsorption orientation identified on mica has the binding cleft between the

17

γC and β C domain (containing parts of the P1 binding site) facing the surface and the P2 and

18

H12 integrin binding sites facing the solution, in line with experimental results showing integrin-

19

mediated interaction between mica-adsorbed Fg and leukocytes 59 and platelets. 9,23

20

We find that the initial adsorption on mica is weak and involves the formation of a small number

21

of contacts. Thus Fg can easily desorb and reorient on the surface. Together with the hinge bending

22

this suggests a significant mobility of Fg on such surfaces. In contrast, the adsorption on graphite

23

is stronger, involving almost an order of magnitude more contacts than on mica, and desorption or

24

reorientation events are rare on the time scales of our simulations. This does not exclude that Fg

25

may be mobile on graphite on time scales much longer than those simulated in the present study.


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The strong interactions between Fg and graphite initiate denaturation already on the comparatively

2

short time scales accessible to our simulations. No indication of a preferred adsorption orientation

3

is found for graphite. These results are in line with previous AFM experimental findings on the

4

relative mobility and denaturation propensity of Fg on mica and graphite surface. 22

5

Acknowledgement

6

The authors thank Prof. H. Heinz for providing the structure of the mica surface and for helpful

7

discussions. SK gratefully acknowledges financial support from the Graduate School Materials

8

Science in Mainz. GS gratefully acknowledges financial support from the Max-Planck Graduate

9

Center with the University of Mainz. We gratefully acknowledge support with computing time

10

from the HPC facility Mogon at the university of Mainz, the Jülich Supercomputing Center and the

11

High performance computing center Stuttgart. This work was partially supported by the German

12

Science Foundation within SFB 1066 (project Q1).

13

Supporting Information Available

14

Document: Supplementary figures, force field parameters. Movie: Example of an adsorption event

15

on mica in which the D-region slides over the surface before beginning to roll.

16

17

18

19

20

21

This material is available free of charge via the Internet at http://pubs.acs.org.

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Molecular Dynamics Simulations of the Initial Adsorption Stages of

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