Charged Surface Regulates the Molecular Interactions of

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Charged Surface Regulates the Molecular Interactions of Electrostatically Repulsive Peptides by Inducing Oriented Alignment Lin Zhang, and Yan Sun Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04308 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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Charged Surface Regulates the Molecular

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Interactions of Electrostatically Repulsive Peptides

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by Inducing Oriented Alignment

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Lin Zhang, Yan Sun*

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Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the

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Ministry of Education, School of Chemical Engineering and Technology, Tianjin University,

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Tianjin 300072, China

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ACS Paragon Plus Environment

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ABSTRACT: Regulation of molecular orientation of charged dipeptides and involved

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interactions by electrostatic repulsion from like-charged surfaces were studied using all-atom

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molecular dynamics simulations. It was found that a charged surface can induce oriented

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alignment of like-charged peptides, and the oriented alignment leads to enhanced electrostatic

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repulsion between the peptide molecules. The findings are consistent with previous experimental

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results about the inhibition of charged protein aggregation using like-charged ion-exchange resin.

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Furthermore, the simulations provided molecular insights into this process, and demonstrated the

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distinct regulation effect of like-charged surfaces on the molecular interactions between peptides

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that possess an electric dipole structure. Both the charged surface and the electric dipole structure

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of peptides were confirmed crucial for the regulation. The research are expected to facilitate the

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rational design of surfaces or devices to regulate the behavior of amphoteric molecules such as

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proteins for both in vivo and in vitro applications, which would contribute to the regulation of

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protein-protein interactions and its application in life science and biotechnology.

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KEYWORDS: charged surface; like-charged peptide, oriented alignment; electrostatic

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repulsion; molecular dynamics simulation

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Langmuir

1.

INTRODUCTION

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Regulation of protein-protein interactions, protein adsorption and protein desorption

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(repulsion) at various surfaces is of fundamental importance, because proteins are the most

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abundant organic molecules in the cell and their interactions at surfaces are crucial in life science

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and biotechnology,1-3 including but not limited to the biotransformation, transportation of matter,

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energy transduction, cell-to-cell interactions and metabolic control.4,5 Molecular behavior of

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proteins adsorbed at surfaces has been extensively studied because of its wide applications

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ranging from material science to chemical biology,6-9 focusing on the orientation and

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conformation of adsorbed proteins and consequent functionality.10-16 For instance, protein

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adsorption at charged surfaces has been extensively investigated using molecular simulation,17-21

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including the adsorption of charged protein to a like-charged surface.

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Besides these adsorbed proteins, proteins repulsed from various surfaces, are also important in

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both life sciences and bioengineering,22-26 but are not well understood. For instance, crowding

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effect22-24 has long been considered effective in regulating protein interactions in vivo to assist

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protein folding. The repulsive interactions between a protein and like-charged crowding agents

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was speculated as a reason for the increased thermodynamic stability of the protein.23,27

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However, possible enhancement on protein-protein interactions under crowded conditions is still

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in dispute and thus an obstacle for the utilization of macromolecular crowding. Investigating the

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influence of various surfaces on protein-protein interactions would be helpful but is usually

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overlooked. Electrostatic forces in solutions of like-charged colloidal particles were investigated,

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and electrostatic attraction was usually more focused.28-31 For electrostatic repulsion, a successful

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regulation on protein-protein interactions using like-charged surfaces in vitro was experimentally

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found32 in our previous work, which facilitated the on-pathway folding of proteins expressed as


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inclusion bodies.33-35 The working mechanism was supposed as that the charged surfaces

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oriented alignment of like-charged protein molecules inhibited protein-protein interactions.

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However, experimental verification of such a mechanism, especially the electrostatic repulsion

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oriented alignment36 was a challenge. Molecular dynamics (MD) simulation, as a powerful tool

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that can offer clear microscopic information in a direct manner,6,37 even the orientational

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alignment of proteins,38 was then used to explore the molecular insights into this process in our

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previous work.39 Standing orientation of lysozyme molecules near a like-charged surface was

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confirmed. However, complicated structure of protein molecules7,14,40,41 limited the investigation

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on the oriented alignment at surfaces and consequent regulation on the molecular interactions.

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Therefore, dipeptides were designed and used instead of protein molecules in this work.

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Chemically, protein molecule is composed of one or more polypeptide chains with complicated

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structures. The dipeptide has much simpler structure than that of a protein, but it can mimic the

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amphoteric feature of proteins, which is expected important for the formation of orientation

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controlled by electrostatic interactions. So the dipeptide was used as a dipole model of protein to

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examine the protein orientation and molecular interactions at like-charged surface. Herein, four

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dipeptides were designed, including KL, LK, LL, and LE, as shown in Figure 1a. Among these

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peptides, both KL and LK are positively charged, but the former has larger electric dipole than

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the latter; LL has several charged sites but is overall neutral; LE is negatively charged. Three

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surfaces were designed for the simulations (Figure 1b), including one neutral, one positively

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charged and one negatively charged. All-atom MD simulations were then performed to examine

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the behavior of the peptides near the surfaces, focusing on the molecular orientations and

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molecular interactions between peptides.


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Figure 1. All-atom models of dipeptides (a) and surfaces (b). Four dipeptides were considered.

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KL has a net charge of +1 at pH 7 because it has 2 positive charges and 1 negative charge. LK

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has also a net charge of +1 contributed by 1 positive charge at the N-terminal. LL is neutral. LE

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has a net charge of -1. Because the dipeptide has heterogeneous charge distribution, the atoms in

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each dipeptide are divided into two groups, one includes all atoms with positive charge and the

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other includes all atoms with negative charge. The center of mass of each group was calculated.

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The equilibrated distance between these two groups was calculated to evaluate the electric

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dipole, as shown below each peptide. All atoms were colored as CPK model and prepared using

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Rasmol.42

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2. MODELS AND METHODS

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2.1. Model Construction. The AA models of dipeptides were constructed and the three

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dimensional structures were determined using CharMM program (http://www.charmm-gui.org/),

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as shown in Figure 1a. The net charge and electric dipole of each dipeptide are shown in Figure

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1a.

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To construct the simulation system in aqueous solution, two dipeptides were randomly placed

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into a simulation box with a size of 6.0 × 6.0 × 6.0 nm3. The solvent molecules were then added.


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The water molecule was treated using SPC model. The system was neutralized by adding Na+ or

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Cl- as counter ions.

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Charged surface was modeled as a neutral planar surface with immobilized positively or

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negatively charged ligands, following the structure of ion-exchange resin.32 The surface is

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composed of repetitive hexagonal unit43,44 with a side length of 0.152 nm (Figure 1b). For the

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ligand, its topology was generated by the Dundee PRODRG 2.5 server (beta)45

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(http://davapc1.bioch.dundee.ac.uk/cig-bin/prodrg_beta).

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distribution were defined based on the parameters of similar structure in literature46 with minor

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modification. Thereafter, ligands were immobilized to the matrix through a C-O-C bridge

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(Figure 1b) with a ligand density of 0.21 mmol/mL according to the experimental data.32 In

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positively/negatively charged surface, each ligand has a protonation state with a net charge of

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+1/-1. Meanwhile, a surface with neutral ligands (with a net charge of zero) was constructed.

Its

charge

group

and

charge

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To construct the simulation system containing charged/neutral surface, the surface was placed

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at the bottom of a simulation box with a size of 7.3 × 6.3 × 6.0 nm3, and an inert surface was

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placed at the top of the box to keep all molecules in the box. Two dipeptides equilibrated in

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aqueous solution were used and put into the simulation box with the equilibrated conformations

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in aqueous solution. That is, the dipeptides were firstly equilibrated in aqueous solution, and then

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the equilibrated structures were used as the initial conformations at surface. Herein, rotation was

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performed to ensure that each dipeptide had a same initial distance of 1.5 nm from the surface.

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Solvent molecules around dipeptides in the equilibration stage were kept except those out of the

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new simulation box. Supplement of solvent molecules was then performed to make correct

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density in the simulation box. The system was neutralized by adding Na+ or Cl- as counter ions.


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These were then placed in the center of a cuboid box of size 7.3 × 6.3 × 100 nm3 for MD

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simulation.

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2.2. Simulation Method. MD simulations in the NVT ensemble were performed using

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Gromacs 4.5.3 package (http://www.gromacs.org/)47,48 and Gromos96 43a1 force field.49 A 2 fs

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time step was used to integrate the equations of motion. The cut-off of the Lennard-Jones (LJ)

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potential was set to 1.2 nm. Standard PME function was used to deal with the Coulomb potential.

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Periodic boundary was used in the x, y and z directions. Neighbor list was updated every 10

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steps. The temperature was controlled at 298.15 K by the Berendsen method with a time constant

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of 0.5 ps. The initial velocities of beads were generated according to the Maxwell distribution at

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system temperature. Simulations were run for 20 ns in aqueous solution firstly, to examine the

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behavior of dipeptides in bulk solution, and to obtain the equilibrated structures to provide the

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initial conformations for the following 5 ns simulation at surfaces, as described in Section 2.1.

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Sixty-four independent simulations were performed for each set of conditions. The snapshots

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were prepared using Rasmol42 (http://www.umass.edu/microbio/rasmol/).

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2.3. Dipeptide Orientation Analysis. The angle (θ) between the direction of dipeptide

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dipole and z axis was calculated to evaluate the orientation of dipeptide, as reported in previous

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work.39,50 When θ=0, the dipole is perpendicular to the surface with the negative pole facing the

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surface. When θ=-90 or 90, the dipole is parallel to the surface (the dipole with positive x

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coordinate is defined as plus, the other minus). θ=180° or -180° indicates a dipole also

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perpendicular to the surface but with the positive pole facing the surface. A parameter P⊥ was

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defined to represent the probability of dipeptide molecule with a θ between -30° to 30° to

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evaluate the dipeptides perpendicular to the positively charged surface. It should be noted that P⊥

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has been normalized by the volume fraction at certain θ in three dimensional space (see


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Appendix S1, Supporting Information). Over negatively charged surface, P⊥ was defined to θ150° instead.

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2.4. Dipeptide Movement Analysis. The z coordinate of the center of mass (com) of the

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dipeptide (zcom) was monitored to depict the location of dipeptide. The distribution probability of

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dipeptide, P(zcom,θ), was then calculated and shown by the filling colors. Herein, P(zcom,θ) was

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calculated based on a statistics in the whole simulation to take all possible angle values. That is,

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the dipeptide molecules with a certain zcom and θ were counted, and the obtained number was

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divided by the total number of dipeptides sampled in the whole simulation to calculate P(zcom,θ).

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Therefore, in the distribution map, at a certain zcom, the range of all possible θ was marked. The

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conformational change of the peptides at the exclusion was evaluated using root mean square

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deviation (RMSD) and radius of gyration (Rg). RMSD representing the structural change of a

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molecule at time t with respect to a reference structure (herein the initial structure is used), was

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calculated using g_rms provided by Gromacs. Rg representing the compactness of a molecule

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was calculated using g_gyrate provided by Gromacs.

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2.5. Molecular Interaction Analysis. The potential energies, including LJ potential and

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Coulomb potential, were calculated using auxiliary program g_energy provided by Gromacs to

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evaluate the molecular interactions between the dipeptide molecules or between the dipeptide

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and the surface. Herein, hydrophobic interaction was considered and included in the LJ potential

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energy. Hydrogen bond was analyzed using auxiliary program g_hbond provided by Gromacs.

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The LJ potential between atoms i and j is calculated by Eq. (1).

VLJ (rij ) =

Cij12 rij12

−

Cij6

(1)

rij6


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rij

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where

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atom types.

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is the distance between atoms i and j;

and

Cij6

are parameters depend on pairs of

The Coulomb potential between atoms i and j is calculated by Eq. (2). VC (rij ) = f

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Cij12

qi q j

(2)

ε r rij

where f is electric conversion factor; ε r is dielectric constant; qi is the charge carried in atom i.

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3. RESULTS AND DISCUSSION

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3.1. Oriented Alignment of Dipeptides over Like-charged Surface. Two dipeptides,

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equilibrated in aqueous solution, were put onto different surfaces with an initial distance of 1.5

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nm to examine the regulation of protein behaviors by surfaces.

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Quantitative description based on a statistics of 64 independent simulations was obtained

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through P ⊥ and zcom values (Section 2.3). Low P ⊥ was observed in the aqueous solution,

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indicating no preferred orientation but only random distribution (Figure S1a in Supporting

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Information). At the neutral surface (Figure 2a), little change of zcom was observed, indicating

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that the peptides randomly moved around their initial positions. The P⊥ values were small with

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random changes, confirming that no oriented alignment was formed. This was confirmed by the

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orientation and location of the peptides in typical snapshots (Figure S2) in a direct manner. No

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obvious movement of the peptides was observed at a neutral surface (Figure S2a); KL moved

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around the initial position and rotated randomly, and no preferred orientation was observed in the

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snapshots. In contrary, at the like-charged surface, the P⊥ of KL changed from initial values to

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approximately 1.0 immediately (within 0.02 ns), confirming the formation of oriented alignment

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where the peptides were perpendicular to the surface with the negative pole facing the surface,


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consistent with the snapshots in Figure S2b (snapshot at 0.02 ns). Slight conformational change

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of the dipeptide was observed, as indicated by the increase of RMSD and Rg values (Figure S3).

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It was attributed to the simultaneous electrostatic attraction at the negative pole of the dipeptide

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and the electrostatic repulsion at the positive pole in the opposite directions, as a result of the

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electric dipole structure. Meanwhile, the peptides were repulsed and then went far away, which

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was consistent with the theoretical calculation of the equilibrated position of electric dipole near

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a charged dot (see Appendix S2). In this process (rapid increase of zcom value), the P⊥ decreased

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and reached an average value of 0.26 at 5 ns with fluctuations, indicating the diminishing of

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oriented alignment following the fast exclusion of the peptides by the like-charged surface.

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Decrease of Rg was also observed (Figure S3), indicating the diminishing of conformational

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change, which is consistent with the protein behavior at desorption stage in chromatography in

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our previous work.13,51-54

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The distribution probability of the peptides was drawn for further evaluation of the orientation

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based on a statistics of 64 independent simulations. Random distribution in aqueous solution was

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observed (Figure S1b), indicated by the wide distribution with low probabilities. Similarly, wide

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distribution of θ with low probabilities was observed at the neutral surface (Figure 2b),

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indicating no preferred θ for the peptides. Meanwhile, the distribution region of the peptides at

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the neutral surface is located at zcom = [0.5,1.5], which is near the initial value of zcom. That is, the

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peptides maintained its initial state without significant change of orientation, consistent with the

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snapshots in Figure S2. However, at the like-charged surface (Figure 2c), a dominant state with

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zcom = [1.5,2.5] and θ = [-20°,20°] was observed, indicating the formation of oriented alignment

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of the peptides. The preferred angle distribution near the like-charged surface located at the

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range corresponding to the oriented perpendicular direction. As compared to the distribution


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probability at neutral surface, an obvious change of peptides to approximately perpendicular

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orientation can be concluded. However, this oriented alignment diminished with increased

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distance from the surface. That is, the convergent angle distribution at small zcom became wider at

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larger zcom (see the distribution in red, Figure 2c). According to the Coulomb potential [Eq. (2)],

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the electrostatic interaction decreases with the increase of distance. Then the diminishing of

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oriented alignment further highlighted the important role of electrostatic interaction from the

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charged surface in this process.

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Figure 2. Molecular behaviors of KL (a-c) and LE (d-f) at a neutral surface and a like-charged

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surface. Time courses of P⊥ and zcom are shown in (a) for KL and (d) for LE, the distribution

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probability near neutral surface represented as a zcom vs θ plot is shown in (b) for KL and (e) for


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LE, and the distribution probability at the like-charged surface is shown in (c) for KL and (f) for

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LE. The distribution probability is shown by the filling colors. Herein, because of the negatively

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charged surface, the P⊥ for LE was defined as the probability of the dipeptide with a θ ≤ -150° or

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≥ 150°, corresponding to an oriented alignment where the dipole is perpendicular to the surface

5

but the positive pole facing the surface. Schematic diagrams composed of representative

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snapshots are provided in the figure top, where the dipoles of the dipeptides, at a direction from

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red (negative pole) to blue (positive pole), is shown for better examination of their orientations.

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The dipole moment is enlarged for clear view.

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For further verification, the molecular behavior of the negatively charged LE at a negatively

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charged surface was examined, as shown in Figures 2d to 2f. The P⊥ of peptides changed to 1.0

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immediately (Figure 2d), indicating the formation of oriented alignment where the peptides were

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perpendicular to the surface with the positive pole facing the surface, similar with the oriented

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alignment of KL over the positively charged surface (Figure 2a). Thereafter, P⊥ decreased with

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the rapid increase of zcom values, and reached an average value of 0.18 at 5 ns with fluctuations,

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indicating the diminishing of oriented alignment following the fast exclusion of peptides by the

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like-charged surface. In the distribution probability of LE at negatively charged surface (Figure

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2f), a dominant state with zcom = [1.7,2.5] and θ ≤ -170° or ≥ 160° was observed, proving the

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formation of like-charged surface oriented alignment.

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3.2. Regulation of Intermolecular Interactions by Charged Surface. Molecular

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interactions between dipeptides were monitored, including the electrostatic interactions,

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hydrophobic interactions and hydrogen bonds. However, the value of LJ potential energy was

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much smaller than that of Coulomb potential energy (see Figure S4), because the LJ potential

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energy decreases much faster with the increase of distance, as shown in Eqs. (1) and (2).


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Hydrogen bond is also only effective within a small distance of about 0.35 nm. Therefore,

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hydrophobic interactions and hydrogen bonds were not discussed herein. The Coulomb potential

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energy and the distance between two dipeptides were used to evaluate the effect of charged

4

surface on the molecular interactions between the two peptides. Herein, △ d=d-d0 was used

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instead of d to avoid the disturbance of initial distance between dipeptides (d0). EC around zero

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was observed with significant fluctuations in aqueous solution (Figure S1c), indicating random

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collision of the peptides in bulk solution. At the neutral surface (Figure 3), EC had significant

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fluctuations near zero but without a definite trend, indicating that random movement did not

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influence the molecular interactions between the peptides. In contrary, at the like-charged surface,

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for both positively charged KL (Figure 3a) and negatively charged LE (Figure 3b), immediate

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increases of EC were observed, indicating that the oriented alignments (Figure 2) indeed

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enhanced the repulsion between the peptides, which was also confirmed by the larger △d than

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that at the neutral surface. However, as the discussion refer to Figure 1, the oriented alignment

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diminished when the dipeptides went away from the surface, because the electrostatic interaction

15

decreased with increased distance according to the Coulomb potential [Eq. (2)]. As a result, the

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enhanced repulsion between the peptides diminished. △ d of KL (LE) dipeptides near like-

17

charged surface decreased and became similar as that above neutral surface at the last part of

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simulation. This further confirmed that the like-charged surface oriented alignment was the

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reason for increased molecular repulsion between the peptides, and this can explain the

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experimental observation of reduced protein aggregation in a suspension with like-charged

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particles.32,35,55,56 From the experimental results, enhanced refolding of charged protein at like-

22

charged surface was confirmed. However, the exploration of molecular mechanism was still a

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challenge using experimental approaches. From the simulation results, the like-charged surface


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oriented alignment and consequent enhancement of electrostatic repulsion between the

2

dipeptides were confirmed. That is, the simulation results can explore the molecular mechanism

3

about the function of charged surface directly. Based on these findings, charged surface can be

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designed and used to induce microscopic oriented alignment of peptides, which leads to the

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enhanced electrostatic repulsion between peptides, and accounted for the macroscopic

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experimental results of reduced protein aggregation by like-charged particles.

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Figure 3. Molecular interactions between KL (a) or LE (b) at a neutral or a like-charged surface,

9

as shown by the time courses of EC and △d. △d is the increase of d as compared to the initial

10

value. Schematic diagrams composed of representative snapshots are provided in the figure top.

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3.3. The Dependence of Oriented Alignment on Electric Dipole. As mentioned above, a

12

like-charged surface was necessary to realize the regulation on protein orientation. Besides this,

13

the electric dipole of peptides may also be crucial. Simulations using other dipeptides with

14

different dipoles and net charges were therefore performed to evaluate the contribution of the


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electric dipole and net charge. LK with the same net charge of +1 as KL but lower charge density

2

was used, together with a neutral dipeptide LL (see Figure 1).

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Figure 4. Molecular behaviors of LK and LL over a positively charged surface, where the time

5

courses of P⊥ and zcom are shown in (a), the distribution probability represented as zcom vs θ plot

6

is shown in (b) for LK and (c) for LL, and the time courses of EC and △d values are shown in

7

(d).

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The P⊥ and zcom were calculated, as shown in Figure 4a. Similar results were observed for LK

9

as those for KL (Figure 2). Oriented alignment was formed immediately and then diminished in

10

the exclusion process of LK. For LL, however, a significant difference was observed. The


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peptides were adsorbed onto the surface very quickly rather than being repulsed, as indicated by

2

the zcom. This was consistent with the theoretical calculation in Appendix S2 (Supporting

3

Information). Obvious oriented alignment of the adsorbed LL molecules was observed at the

4

surface. That was attributed to the heterogeneous charge distribution of LL. That is, LL should

5

also be considered as an electric dipole with distinct negatively charged part (negative pole) and

6

positively charged part (positive pole). The attraction of negative part at surface induced the

7

formation of oriented alignment. Moreover, LL has an overall neutral charge, so the attraction

8

became stronger due to smaller distance as compared with the repulsion of positively charged

9

part (positive pole), leading to the adsorption rather than repulsion of the whole LL molecule.

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The distribution probability was calculated to evaluate the orientation of the peptides at the

11

charged surface based on a statistics of 64 independent simulations, as shown by the filling

12

colors in Figures 4b and 4c. For LK, a dominant distribution located in the region of θ = [-30°,

13

30°] was observed, indicating the formation of a perfect oriented alignment where the peptides

14

were perpendicular to the surface with the negative pole facing the surface. More converged state

15

around θ = 0° was observed for LL with a small zcom, indicating the formation of oriented

16

alignment on the surface as well as the dominant attraction. The more converged state further

17

confirmed the enhanced electrostatic attraction due to the small distance from the surface.

18

The Coulomb potential energy and the distance between two dipeptides were monitored to

19

examine the contribution of charged surface on inhibiting the aggregation of the peptides, as

20

shown in Figure 4d. For LK, rapid increase of △d was observed, followed by an immediate

21

increase of EC, indicating the oriented alignment indeed enhanced the repulsion between the

22

peptides. As compared to KL (Figure 3a), however, the increase of EC was smaller. This clearly

23

indicates that the smaller dipole resulted in a weaker enhancement of repulsion between the


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1

peptides at like-charged surface. For LL, the increase of EC was also observed, but without

2

significant increase of △d, because the peptides were adsorbed onto the surface and could not

3

move freely (see zcom in Figure 4a). Similar results were obtained for the positively charged KL

4

at a negatively charged surface (see Figure S5).

5

Therefore, the results indicated that the net charge of protein determines the adsorption or

6

repulsion at a charged surface. Charged protein is repulsed from a like-charged surface, such as

7

the repulsion of positively charged KL by a positively charged surface, or negatively charged LE

8

by a negatively charged surface (Figure 2). In contrary, protein without effective repulsion by the

9

surface will be adsorbed, such as the adsorption of neutral LL on a positively charged surface

10

(Figure 4), or positively charged KL at a negatively charged surface (Figure S5). The

11

dependence of adsorption or repulsion on the net charge of protein is consistent with the

12

theoretical calculation in Appendix S2 (Supporting Information). Moreover, the regulation of

13

intermolecular interactions of adsorbed peptides was different from that for repulsed peptides.

14

Because the electrostatic interaction decreased with increased distance according to the Coulomb

15

potential [Eq. (2)], the diminishing of regulation was observed during the excluding process of

16

repulsed peptides, as the discussion refer to Figure 1. For the adsorbed peptides, the maintained

17

small distance from the surface caused large electrostatic interaction and strong regulation. For

18

example, LL adsorbed at the surface make it obtain a large P ⊥ above 0.6 and a large △ d,

19

indicating a good spatial isolation of peptides. The spatial isolation of proteins on ion-exchange

20

chromatography (IEC) was widely described in experimental results, and was considered as a

21

driving force for the protein refolding in IEC.57-59 Protein adsorption driven by electrostatic

22

attraction from the IEC resin is the key to separate the bound protein molecules and helpful for

23

the protein refolding.


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1

Moreover, the results indicated that the electric dipole of a protein is crucial for the formation

2

of charged surface oriented alignment. A protein with heterogeneous charge distribution can

3

possess preferred orientation at a charged surface. As evidenced by the results in the present

4

study, all the four dipeptides, KL, LK, LL and EK, can form oriented alignment at a charged

5

surface due to their electric dipoles, regardless of being adsorbed or repulsed. The attraction from

6

the surface to the part of a dipeptide with opposite charge is the determinant for the preferred

7

adsorption sites. For example, the negative pole of LL determined the preferred orientation of LL

8

and consequent oriented alignment. The adsorption of protein on a charged surface due to its

9

heterogeneous surface has been extensively reported in the simulation results.17-21 The preferred

10

adsorption sites and orientation have been discussed. The simulation results of LL on a charged

11

surface, featured by the preferred adsorption sites and oriented alignment, are consistent with the

12

results reported in literature.57-59 Moreover, the regulation of molecular interactions between

13

peptides by the oriented alignment was highlighted herein, which accounted for the experimental

14

observation about the inhibition of protein aggregation by like-charged resins.32 This may be the

15

reason for protein folding rather than aggregation in crowded conditions, which can interpret the

16

crowding effect. In crowded conditions, proteins are always near various surfaces (including

17

various charged surfaces) provided by the crowding agents. The induced oriented alignment of

18

protein by such charged surfaces can be expected due to the heterogeneous charge distribution

19

(electric dipole) of protein molecules, which would then enhance the repulsion between proteins

20

and thus help enhance the inhibition of their aggregation and improve the on-pathway protein

21

folding.

Page 18 of 31

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4. CONCLUSIONS

2

In summary, charged surfaces can induce the formation of oriented alignment of peptides,

3

leading to enhanced electrostatic repulsion between peptides or proteins, which is considered as

4

the reason for the inhibition of aggregation and the enhancement of on-pathway protein folding.

5

The oriented alignment of peptides diminished with increasing distance from the like-charged

6

surface, and can be enhanced by increasing electric dipole of the peptides. The simulation results

7

explained the experimental observation of reduced protein aggregation in a suspension with like-

8

charged particles, and confirmed the like-charged surface oriented alignment and consequent

9

enhanced molecular repulsion between the peptides at molecular level. These results have

10

explored the working mechanism of charged surfaces on the regulation of molecular interactions

11

between peptides, which would be helpful for interpreting the crowding effect in vivo and the

12

enhanced on-pathway folding of high concentration proteins by like-charged additives in vitro.

13

The research will thus facilitate the rational design of surfaces or devices for regulating the

14

interfacial behaviors of protein molecules both in vitro and in vivo.

15 16

ASSOCIATED CONTENT

17

Supporting Information.

18

Appendix about the derivation of volume fraction in three dimensional space, appendix about the

19

calculation of the equilibrated position of electric dipole near a charged dot, molecular behaviors

20

of KL and LE in aqueous solution, snapshots of two KL dipeptides over a neutral surface and a

21

charged surface, LJ potential energies of KL and LE dipeptides at a like-charged surface, the

22

time courses of RMSD and Rg of KL and LE dipeptides at a like-charged surface, and molecular


19

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1

behaviors of KL at a negatively charged surface. This material is available free of charge via the

2

Internet at http://pubs.acs.org.

3

AUTHOR IMFORMATION

4

Corresponding author

5

*Tel & Fax: +86 22 27403389; E-mail addresses: [email protected] (Y. Sun).

6

Author Contributions

7

The manuscript was written through contributions of all authors. All authors have given approval

8

to the final version of the manuscript.

9

Notes

10

The authors declare no competing financial interest.

11

ACKNOWLEDGMENTS

12

This work was supported by the Natural Science Foundation of China (Nos. 91534119,

13

21236005, 21376173 and 21621004) and the Innovation Foundation of Tianjin University.

14 15

REFERENCES

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Table of contents only


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Figure 1 64x23mm (300 x 300 DPI)


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Figure 2 134x102mm (300 x 300 DPI)


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Figure 3 94x49mm (300 x 300 DPI)


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Figure 4 133x99mm (300 x 300 DPI)


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TOC 56x37mm (300 x 300 DPI)


Charged Surface Regulates the Molecular Interactions of

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