Article pubs.acs.org/JPCB
Environmental Effect on Surface Immobilized Biological Molecules Zunliang Wang,†,‡ Xiaofeng Han,† Nongyue He,*,† Zhan Chen,*,‡ and Charles L. Brooks, III*,‡ †
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Si Pai Lou 2, Nanjing 210096, China ‡ Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States ABSTRACT: Our recent sum frequency generation (SFG) vibrational spectroscopic experiment (J. Phys. Chem. B 2014, 118, 2904−2912) showed that immobilized antimicrobial peptide cecropin P1 (cCP1) on a self-assembled monolayer (SAM) surface via N-terminus exhibited significantly different conformational and/or orientational behaviors when exposed to pure water vs a 50% (v/v) 2,2,2-trifluoroethanol (TFE)/water mixture. Meanwhile, our recent molecular dynamics (MD) simulations (J. Phys. Chem. B 2014, 118, 5670−5680) further revealed that the immobilized cCP1 via N-terminus in pure water largely adopts an overall bent structure lying down on the SAM surface, consistent with the SFG observation. Here, MD simulations were performed on the immobilized cCP1 on a SAM surface via N-terminus while in contact with a 50% (v/v) TFE/water mixture to further investigate the effects of environment (water vs TFE/water mixture) on the interfacial structure and orientation of immobilized peptide. The simulation results demonstrated that the immobilized cCP1 on the SAM surface via the N-terminus with two different starting states with different orientations and conformations, when exposed to a 50% (v/v) TFE/water mixture, was eventually able to maintain a linear α-helical structure, standing upright on the SAM surface. Taken with the corresponding SFG observation, our simulation results indicate that the conformational behavior of the immobilized peptide is mediated by the local hydrophobic environments resulting from the TFE aggregation around the peptide. Such knowledge can be used to regulate the surface conformation and functionality of immobilized peptides via changing surrounding chemical environments (e.g., TFE cosolvent), which is important for the microbial detection and killing based on surface-immobilized antimicrobial peptides.
1. INTRODUCTION Natural antimicrobial peptides are important in the immune system, protecting the host from microbial invasion.1−4 They can selectively bind to and disrupt the microbial cell membranes to kill microbials. Due to their strong affinity for microbial membranes, surface-immobilized antimicrobial peptide arrays can be used for rapid detection of microbial targets including bacterial and viral pathogens.5−18 For such detection arrays, stable surface structure and optimized orientation of immobilized peptides are crucial for their surface activity.19−21 Understanding such peptide structure and orientation aids in the rational design and development of peptide arrays with improved performance in microbial capture and death. Besides the physicochemical properties of the substrate surface, the conformational/orientational behavior of immobilized peptides on surfaces must be also mediated by the complex interactions between the amino acids in the peptide and the surrounding environment. Effects of chemical environment on peptide conformation and stability have been extensively investigated, and 2,2,2-trifluoroethanol (TFE) is often used as a cosolvent in peptide and protein conformational studies, due to its ability to promote and reinforce the α-helical secondary structure.22−25 © XXXX American Chemical Society
Many experimental methods have been used to characterize the surface structure of immobilized peptides, including scanning tunneling microscopy (STM),26 electrical impedance spectroscopy (EIS),27 X-ray photoelectron spectroscopy (XPS),27 Fourier transform infrared (FTIR) spectroscopy,27 circular dichroism (CD) spectroscopy,4 and sum frequency generation (SFG) vibrational spectroscopy.28−61 Among these techniques, SFG spectroscopy stands out as a powerful characterization technique with submonolayer sensitivity in probing surface-immobilized peptides at the solid/liquid interface at the molecular level and in situ. We recently studied the surface immobilized antimicrobial peptide cecropin P1 (CP1, sequence SWLSKTAKKLENSAKKRISEGIAIAIQGGPR) using SFG.62 It was found that surface-immobilized CP1 via an N-terminally modified cysteine (cCP1) exhibited a much weaker SFG amide I signal in pure water but a very strong α-helical SFG amide I signal in the 50% (v/v) 2,2,2trifluoroethanol (TFE)/water mixture.62 The observed difference between the SFG spectra in the two different solvent Received: August 24, 2014 Revised: September 27, 2014
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investigate the TFE-induced interfacial conformational transition of the immobilized peptide. The immobilized peptide cCP1 monomer consists of 31 amino acid residues, with an additional cysteine residue at the N-terminus used to covalently bind the peptide to the maleimide group on the SAM. The cCP1 monomer has positively and negatively charged N-terminal NH3+ and Cterminal COO− groups, respectively. In the peptide, Lys (K) and Arg (R) residues are positively charged, Glu (E) is negatively charged, and other residues are neutral, giving each of the cCP1 molecule a total charge of +5. In Simulation I, the initial tilt angle of the α-helical peptide vs the SAM surface normal was ∼10°. The SAM was modeled with a monolayer containing 457 silane-EG 4 -maleimide [(OH) 3 Si(CH 2 ) 2 NH-CONH-EG 4 (CH2)2N(CO)2C2H2] chains on a silica substrate at a density of 4.5 chains per nm2. This is consistent with the experimental condition of 4−5 chains per nm2 for a full coverage of SAM on silica.78−80 The initial SAM structure was constructed using the CHARMM program.81−83 The maleimide group of the EG4SAM was modeled using the CHARMM27-compatible force field parameters developed by Imhof and co-workers.84 The force field parameters for each EG4 unit were directly adopted from the CHARMM27 force field.85,86 All the silicon atoms and the hydroxylated α-quartz (011) slab used for the silica substrate were modeled using the CHARMM27-compatible silica force field developed by Lopes et al.87 To build the solvent box of the TFE/water mixture, we started from a pre-equilibrated TIP3P88 water box with a side length of 19 Å produced by the CHARMM program.81−83 To obtain the 50% v/v concentration, we randomly added TFE molecules into the water box, where the water molecules overlapping within 2.0 Å around the TFE molecules were removed. The final molecular number ratio of water/TFE was 4:1 (216 water and 54 TFE molecules), producing a density of 1.20 g/mL corresponding to the reported experimental data.89 The new solvent box containing the 50% (v/v) TFE/water mixture had a dimension of 23 × 23 × 23 Å3. First, the solvent box was equilibrated for 3 ns in the NVT ensemble at 300 K, and then continued for 3 ns in the NPT ensemble. For all simulations in this study, the TFE molecules were modeled using the CHARMM-27 compatible force field developed by MacKerell group.90 Both simulation systems were solvated in the equilibrated 50% (v/v) TFE/water solvent box noted above. Each solvated system was then neutralized by adding 5 Cl− ions. For Simulation I, there were 2580 TFE and 15 566 water molecules. For Simulation II, there were 2685 TFE and 16 174 water molecules. Each simulation system was then subjected to an energy minimization procedure to remove unfavorable contacts. This procedure first involved solvent minimization using 100 steps of the steepest descent (SD)81 and 1000 steps of adopted basis Newton−Raphson (ABNR)81 minimization, with the silicon atoms fixed and the non-hydrogen atoms of the solutes (peptide and SAMs) restrained with a harmonic force constant of 40 (kcal/mol)/Å2. This was followed by five similar rounds of the SD-ABNR minimizations, during which the harmonic restraints on the heavy atoms of the solutes were then gradually reduced to zero. 2.2. Simulation Protocol. After energy minimization, all simulation systems were then gradually heated from 0 to 300 K in increments of 25 K for every 5 ps, while harmonically restraining the peptide backbone atoms and the SAM to their
environments indicates that in pure water, the immobilized cCP1 via N-terminus is likely to adopt a random coil or a lyingdown α-helical structure on the self-assembled monolayer (SAM) surface, whereas after the addition of 50% TFE to the water solvent, the immobilized peptide may refold to an α-helix with an upright orientation on the SAM surface. Although this SFG observation can capture the TFE-induced difference in the conformational/orientational behavior of surface-immobilized peptides, the details of the structural and orientational changes as well as dynamics of the immobilized peptides has not been completely understood yet. Molecular dynamics (MD) simulations can provide an important complement and more detailed information and have been widely used in understanding complex surface conformational behavior of proteins and peptides.63−72 Using MD simulation methods, we and other research groups have investigated the stabilizing effect of TFE on the α-helical structure of peptide.73−76 Almost all of these studies mainly focused on the peptide conformational behavior in bulk solutions. The effect of TFE on the structure and orientation of the peptides immobilized on surfaces is still unclear. Our recent MD simulation study on the surface-immobilized peptide in pure water demonstrated that the N-terminally immobilized cCP1 adopted an overall bent structure, lying down on a SAM surface.77 Such structural and orientational behavior is well correlated with the SFG observation that very weak SFG signals were detected from the immobilized cCP1 in pure water.62 The strong hydrophobic interaction of the free Cterminal amino acid residues with water is the dominant driving force leading to the surface parallel orientation of immobilized cCP1 and its conformational transition from an surface normal linear structure to a lying-down bent structure. Here, we further use all-atom MD simulations to investigate the effect of TFE on the interfacial structure and orientation of surface-immobilized cCP1. The structural and orientational dynamics of the immobilized peptide were derived from the MD simulations and compared to the available SFG experimental data. In particular, the local concentration of the TFE molecules surrounding each peptide residue was examined. To further understand the interaction of TFE with the surface-immobilized peptide, we analyzed the TFE structural arrangement around the peptide using the radial distribution functions. The underlined molecular mechanism of the TFE-induced structural and orientational changes of surface-immobilized peptide were also elucidated by such analysis.
2. METHOD 2.1. System Setup. Current MD simulations of surfaceimmobilized peptide cCP1 were performed in a 50% (v/v) TFE/water mixture at 300 K. In this work, two initial conformational/orientational states were considered: an αhelical structure for the entire peptide standing upright on the SAM surface (Simulation I), and a bent structure lying down on the SAM surface (Simulation II). These two states were directly adopted from the starting (at 0 ns) and the final (at 60 ns) snapshot structures of the previous MD simulations carried out in pure water, respectively.77 The MD simulation (Simulation I) that started with the α-helical peptide standing upright on the SAM surface was used to evaluate the effect of TFE on the structural and orientational stability of the surface-immobilized peptide. The MD simulation (Simulation II) that started with the bent structure lying down on the SAM surface was used to B
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initial positions. The heating process allowed the initial relaxation of water and TFE molecules around the peptide and the SAM surface. Following this, a full MD run of 50 ns was performed for each simulation system without any restraints on the peptide. All MD simulations in this work were performed with the NAMD2.8,91 using the CHARMM27 force field.85,86 Periodic boundary conditions92 were applied for both simulations. For Simulation I, the simulation box had a dimension of 94 × 94 × 129 Å3, containing 108 178 atoms. For Simulation II, the simulation box had a dimension of 96 × 96 × 130 Å3, containing 110 858 atoms. All of the MD trajectories were generated using the NVT ensemble at 300 K. During both MD simulations, all silicon atoms were fixed, and all covalent bonds involving hydrogen atoms were constrained using the RATTLE method.93 The velocity Verlet method94 was used to integrate Newton’s equations with a time step of 2.0 fs. The short-range VDW interactions were calculated by the switch function with a twin range cutoff between 12 and 14 Å. The long-range electrostatic interactions were calculated by the force-shifting function at a cutoff distance of 14 Å. 2.3. Data Analysis. In both simulations, the coordinates and velocities of the MD trajectories were saved every 2 ps for the analysis of the MD trajectory. The root-mean-square deviations (RMSDs) of the peptide backbone atoms enable the structural changes to be examined throughout the MD simulation. The RMSD in this work was calculated by
LTC(d) =
g (r ) =
3. RESULTS AND DISCUSSION 3.1. Effect of TFE on the Stability of the SurfaceImmobilized α-Helical Peptide in Simulation I. To investigate the stabilizing effect of TFE on the conformation and orientation of surface immobilized α-helical cCP1 in a 50% (v/v) TFE/water mixture, the root mean-square deviations (RMSDs) of the backbone atoms of cCP1 in Simulation I were calculated as a function of simulation time (Figure 1a). It is clearly shown that in the TFE/water mixture the immobilized cCP1 largely remains in its starting α-helical conformation because of the very small RMSD change around 2.5 Å during the entire 50 ns MD simulation. While in water, as demonstrated by our previous MD simulations, the RMSD value of cCP1 exhibits a large increase suggesting a more substantial structural change in the peptide because of its strong C-terminal hydrophobic fluctuation.77 Moreover, to observe the local motion of each amino acid residue, the root-mean-square fluctuation (RMSF) of the backbone Cα atom of every peptide residue was calculated as a function of time throughout the 50 ns simulation (Figure 1b). It is evident that almost every amino acid residue in cCP1 experiences a small structural fluctuation with a RMSF value below 2.0 Å (except for the two residues at the C-terminus which have much larger values), indicating that the α-helical conformation of the immobilized cCP1 in TFE/ water mixture is well maintained despite the C-terminal slight fraying. To characterize the compactness of the entire peptide, the time evolution of the radius of gyration (Rg) of the cCP1 is presented (Figure 1c). It is clearly demonstrated that the Rg of the peptide constantly has a large value of 14 Å throughout the simulation. This means that the surface-immobilized cCP1 well maintains a linear structure in the TFE/water mixture, which is different from its compacted bent structure on surfaces in pure water as demonstrated in our previous simulation study.77 Furthermore, we examined the orientational change of the cCP1 by calculating the peptide tilt angle (τ) with respect to the SAM surface normal as a function of time and its distribution (Figure 2a,b). It is obviously shown that the tilt angle τ remains quite stable and only varies inside a small range of 0−10° (Figure 2a). This indicates that cCP1 more or less maintains its initial upright orientation with respect to the SAM surface during the simulation. For immobilized cCP1, although the initial conformation is the same, the MD trajectories of the
N
where ri,t is the current position of atom i at time t, and ri,0 is its initial position at time t = 0 after eliminating the rotation and translation motions; N denotes the total number of atoms of the immobilized peptide backbone. The radius of gyration (Rg) of the peptide backbone was used to evaluate the overall compactness of the peptide, which is defined as the root-mean-square distance from the center of mass of the peptide to every peptide backbone atom: N
∑i = 1 mi(ri − rCOM)2 N
∑i = 1 mi
where N is the number of atoms, mi and ri are the mass and position of atom i, and rCOM is the center-of-mass position of the peptide. The surface orientational behavior of the immobilized peptide can be characterized by the tilt angle (τ). τ is the angle between the principal axis (h) of the peptide and the SAM surface normal (which is along the z-axis), which can be defined as τ = cos−1
V N (r ,dr )mean NANB 4πr 2 dr
where r is the distance between atoms A and B. N(r,dr)mean is the average number of atom A between r and r + dr from atom B, which can be derived from a MD trajectory. NA and NB are the numbers of atom A and atom B, respectively. Here, using the RDF for TFE around peptide permits the structural arrangement of TFE around a peptide to be evaluated.
N
Rg =
× 100
wat where Vtfe mol = 0.07 and Vmol = 0.019 (L/mol), denoting the average occupied volumes of a TFE and water molecule, respectively. The radial distribution function (RDF) for two sets of atoms (e.g., A and B) can be used to characterize the occurrence probability of atom A around atom B at a given distance. The RDF was calculated according to the following equation:
∑i = 1 (ri , t − ri ,0)2
RMSD =
tfe Vmol Ntfe(d) tfe wat VmolNtfe(d) + Vmol Nwat(d)
h·ẑ |h|
where principal axis vector h is calculated as the eigenvector with the smallest eigenvalue of the inertia tensor of the peptide Cα atoms and ẑ is the unit vector along the z-axis direction which is perpendicular to the SAM surface.77,95,96 The local TFE concentration (% v/v) (LTC) around every amino acid residue was calculated from the cumulative number of TFE (Ntfe(d)) and water (Nwat(d)) molecules present within a distance of d from the backbone atom Cα using the following formula:74,76 C
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Figure 2. (a) Tilt angles of CP1 versus the surface normal in Simulation I as a function of simulation time and (b) its corresponding distribution throughout the 50 ns MD simulation.
sharply increased to 5 Å in the 8−12 ns simulation time range, then it fluctuated around 5 Å with an amplitude of 2 Å during the period of 12−30 ns. After that, it rapidly reached a value of 8 Å at 35 ns and remained steady at 8 Å for the last 15 ns of the simulation time. This result suggests that cCP1 underwent substantial conformational changes on the SAM surface during the 50 ns MD simulation. The average RMSF per amino acid shows that the overall movement of C-terminal residues is much larger than that of the N-terminal residues. Moreover, in the 50 ns period of time, the Rg of cCP1 gradually increased from 10 to 13 Å (Figure 3c), indicating that the immobilized cCP1 tends to extend its initial bent structure to a more linear structure. In addition, the tilt angle of cCP1 gradually decreased from 90° to 50° versus the surface normal (Figure 4), indicating that the peptide tends to orient itself upright with its structure gradually refolding to a linear conformation in the presence of TFE. This trend of conformational transition is well reflected by the representative snapshots from the whole simulation (Figure 5). For cCP1, the population of local secondary structure per amino acid in every 10 ns simulation time except for the Nterminally modified cysteine is presented in Figure 6. In the TFE/water mixture, many amino acid residues adopt a 100% local α-helix conformation during the entire 50 ns simulation time. The C-terminal segment (QGGPR) is less stable, and in the last 10 ns simulation time, it tends to form a turn structure. The local α-helix conformation of the central residue Ser-13 gradually increased its population from 80% to 100% concomitantly with its coil population gradually decreasing to zero, leading to the formation of a linear helical conformation for the entire peptide. This result shows that TFE not only facilitates the stability of the α-helix immobilized on surfaces but also can promote the immobilized peptide to gradually form a linear standing up structure. 3.3. TFE Concentration around the Surface-Immobilized Peptide in Simulation II. To quantify the contacts and
Figure 1. (a) Root mean-square deviations (RMSDs) of the backbone atoms of cCP1 in Simulation I as a function of simulation time. (b) Root-mean-square fluctuations (RMSF, a measure of the average atomic mobility) of the backbone Cα atom of each amino acid residue in cCP1 in Simulation I, averaged over the whole 50 ns MD simulation. (c) Time evolution of the radius of gyration (Rg) of the backbone atoms of cCP1 in Simulation I.
cCP1 generated in the presence of TFE demonstrated completely different structural and orientational behavior of surface immobilized peptide from that generated in pure water.77 This significant difference also indicates that TFE contributes to reinforce the structural and orientational stability of an α-helical peptide immobilized on the SAM surface, which also well agrees with our recent SFG experimental result: The SFG spectra collected from cCP1 showed a much weaker amide I signal in water, whereas a very strong SFG α-helical amide I signal was generated when immersed in a 50% (v/v) TFE/water mixture.62 Such an observed difference in SFG spectra suggests that immobilized cCP1 via the N-terminus tends to maintain a more bent structure lying down on the SAM surface in water but can rapidly regain a more linear αhelical structure standing upright on the SAM surface after the addition of 50% TFE to the contacted water solution. To further reveal conformational dynamics of immobilized cCP1, we performed another 50 ns MD run in Simulation II. 3.2. TFE-Induced Conformational Transition of the Surface-Immobilized Peptide in Simulation II. To further understand the conformational dynamics of surface-immobilized cCP1, the time evolution of RMSD and the RMSF per residue were calculated in Simulation II starting from a different initial state (with peptide in a bent lying down state) to observe the overall time-dependent and local structural variations of the peptide, as shown in Figure 3a,b. For cCP1, the RMSD value D
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Figure 3. (a) Root mean-square deviations (RMSDs) of the backbone atoms of CP1 in Simulation II as a function of simulation time. (b) Root-mean-square fluctuations (RMSF, a measure of the average atomic mobility) of the backbone Cα atom of each amino acid residue in CP1 in Simulation II, averaged over the whole 50 ns MD simulation. (c) Time evolutions of radius of gyration (Rg) of the backbone atoms of CP1 in Simulation II.
Figure 4. (a) Tilt angles of cCP1 versus the surface normal in Simulation II as a function of simulation time. (b) Tilt angle distribution in the whole 50 ns simulation and (c) the last 10 ns simulation.
specific. Our previous simulation result showed that for the surface immobilized cCP1, its bent lying-down structure in pure water is mainly caused by the hydrophobic interactions of the central and C-terminal residues with the surface.77 Thus, combined with the structural analyses as presented above, we believe that the tight association of TFE around the immobilized peptide should also be correlated with its linear α-helical structure and standing upright surface orientation. To further confirm this, we examined the interactions of TFE molecules with the peptide, especially with the hydrophobic residues such as Leu, Ala, and Ile. 3.4. Interaction of TFE with the Surface-Immobilized Peptide in Simulation II. First, radial density distribution functions (RDFs) were calculated to observe the structural arrangement of the solvent (TFE and water) molecules around the peptide. The left panel of Figure 8a shows the RDF profile of the carbon in the CF3 group of TFE and the water oxygen atom with respect to the Cα atom of the peptide. In the case of water oxygen, the RDF profile shows no obvious peak, indicating a less organized water structure surrounding the peptide. Conversely, a relatively strong hydrophobic interaction was observed between the carbon atom in the TFE CF3 group and the peptide backbone Cα atom within a distance of 4.5 Å. This difference means that the number of TFE molecules near the peptide is significantly higher than that of water indicating
interactions of TFE molecules with the surface immobilized peptide, the LTC per residue of CP1 has also been evaluated in two temporal windows including 0−10 and 40−50 ns, as shown in Figure 7a,b, respectively. The LTC per amino acid in CP1 was evaluated by calculating the relative numbers of TFE and water molecules within a solvation shell with a 6 Å surrounding each residue. In the first 10 ns simulation, the average LTC around each residue is 62% (v/v), whereas in the last 10 ns simulation, it increased to 74% (v/v). This temporal difference in LTC shows that TFE gradually aggregates around the peptide with the increase of simulation time. Additionally, it was found that the average LTC around the nonpolar (hydrophobic) residues (Leu, Ala, Ile, Gly, and Trp) is 83% (v/v), whereas the average LTC around the polar hydrophilic residues is 67% (v/v), indicating that TFE molecules have much stronger solvation preferences for the hydrophobic residues. Moreover, all highest values of LTC (higher than 90% (v/v)) correspond to the most hydrophobic (nonpolar) residues: Ala-7, Leu-10, Ala-14, Ile-18, Ile-22, and Ala-25, further suggesting that the preferred aggregation of TFE is determined by the specific hydrophobic nature of each amino acid. This preferred aggregation behavior has been observed in other studies using MD simulations in bulk solvent74,76 and reveals that aggregation of TFE around the peptide is residueE
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Figure 5. Representative snapshots of immobilized cCP1 exposed to 50% (v/v) TFE/water mixture (Simulation II). The backbone of cCP1 is represented with green NewCartoon style. All the snapshots are visualized using VMD1.9.99
Figure 8. Radial distribution functions, averaged over the last 10 ns of the simulation time in Simulation-II for (a) (left panel) CF3 carbon of TFE (red line) and water oxygen (blue line) toward backbone Cα atom of CP1 and (right panel) TFE oxygen and water oxygen (red line), terminal (OH) hydrogen of TFE and water oxygen (blue line); (b) (left panel) CF3 carbon of TFE toward hydrophobic (nonpolar) side chain (red line) and hydrophilic (polar) side chain (blue line) of CP1 peptide and (right panel) water oxygen toward hydrophobic (nonpolar) side chain (red line) and hydrophilic (polar) side chain (blue line).
Figure 6. Secondary structure population of each amino acid residue in CP1 in Simulation II calculated with STRIDE (40) (α-helix (red), turns (green), coil (blue), and 3-10 helix (violet)) in the temporal window of (a) 0−10 ns, (b) 10−20 ns, (c) 20−30 ns, (d) 30−40 ns, and (e) 40−50 ns MD simulations in 50% (v/v) TFE/water mixtures.
nearby TFE molecules are much closer to the peptide compared to water, which is also consistent to the above LTC analysis. To further examine the interaction between the TFE molecules and the water molecules surrounding the peptide, the RDF of the distance between the water oxygen and TFE oxygen within a distance of 6 Å from the peptide backbone Cα atom was presented in the right panel of Figure 8a. The first peak probability of the oxygen−oxygen distance occurs at a short distance of 2.75 Å, indicating strong hydrogenbonding interaction between TFE and water molecules, which was accompanied by the occurrence of the first peak at 1.85 Å between the water oxygen and the hydrogen in the TFE hydroxyl group. To examine the overall orientation of TFE with respect to the peptide residues, we calculated the RDF of the CF3 carbon and oxygen of TFE toward the side chains of the hydrophobic and hydrophilic residues, as shown in the left panel and right panel of Figure 8b, respectively. It is clearly shown that the TFE CF3 carbon has a much higher distribution density around the hydrophobic peptide side chains, and a lower distribution density around the hydrophilic peptide side chains (Figure 8b, left panel), indicating that the TFE molecule interacts more favorably with the hydrophobic (nonpolar)
Figure 7. Local TFE concentration (LTC) within a distance of 6 Å from the Cα atom in each residue of CP1 exposed to the 50% (v/v) TFE/water mixture in Simulation II, averaged over (a) the first 10 ns and (b) the last 10 ns MD trajectories, respectively. Each Bar denotes the LTC standard deviations.
F
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In all RDF profiles, the sharp peaks of the CF3 carbon, CH2 carbon, and oxygen of TFE versus the CH3 carbon are located at 4.5, 5.5, and 6.5 Å, respectively. Accordingly, the CF3 carbon around the Ala, Ile, and Leu also shows a more organized structure compared to the CH2 carbon and oxygen atom of TFE. This result strongly suggests that the TFE is preferentially oriented with the CF3 group pointing to the peptide hydrophobic group. The structure and orientation of TFE in the solvation shell supports the role of its aggregation around the peptide to stabilize the α-helix conformation. This also supports the peptide structural transition from bent to linear in response to the local environmental condition changes. The N-terminally immobilized cCP1 in water has primarily a bent lying down structure. After the addition of TFE to water, the cCP1 molecule tends to transform to a linear α-helical structure for the entire peptide with an upright orientation on the surface, in agreement with our recent SFG experimental result. Our simulation results indicate that the preferred orientation of TFE toward the nonpolar residues can largely promote TFE aggregation around these residues, which further confirms above LTC analysis. Figure 10 shows the final snapshot after 50
amino acids. In the right panel of Figure 8b, the TFE oxygen obviously shows a less organized structure near both hydrophobic and hydrophilic side chains because the RDF profile does not exhibit any obvious peak, indicating that the oxygen of TFE is farther away from the peptide than the terminal carbon of TFE. In addition, the TFE oxygen density around the hydrophobic side chains is on average larger than that around the hydrophilic side chains, suggesting that the TFE molecules adopt a consistent orientation pointing toward the hydrophobic and hydrophilic residues. Thus, we can determine the orientation of the TFE molecule from the above discussion that the hydrophobic CF3 group pointing toward the peptide and the OH group pointing away from the peptide. Because the acidity of the OH group in TFE is increased by the hydrophobic electron-withdrawing trifluoromethyl group, TFE is a good donor but a poor acceptor for hydrogen bonds. Furthermore, in the local solvation shell surrounding the peptide, the hydrogen-bonding association of the TFEhydrogen to the water-oxygen can also bring the water-oxygen closer to the TFE-oxygen. Thus, the water-oxygen in the peptide solvation shell largely points to the peptide. To further explore the nonpolar interactions of TFE with the hydrophobic residues, the RDF profiles for terminal (CF3) carbon, the central carbon and the oxygen of TFE to the CH3 carbon of the Ala, Ile, and Leu residues (corresponding to the highest LTC values) are presented in Figure 9a−c, respectively.
Figure 10. Snapshot after the 50 ns MD run in Simulation II. The backbone of CP1 is represented with green NewCartoon style. Only the solvent molecules (TFE and water) within a distance of 10 Å from the peptide backbone are shown. TFE molecules are represented with red licorice style and water molecules are represented with blue points style. The bottom SAM surface is represented with lines style, colored with its atom nature. The snapshot is visualized using VMD1.9.99
ns of simulation time in Simulation II. It is evident that TFE molecules largely aggregate around the peptide, reducing the interaction of water with the peptide.
4. CONCLUSION In the present study, we performed molecular dynamics simulations to investigate the structural and orientational changes of N-terminally immobilized peptide cCP1 exposed to 50% (v/v) TFE/water mixture. Analyses of the MD trajectories starting from two peptide initial structures, a standing-upright α-helix and a bent structure with a lying down orientation, showed that TFE is able to not only reinforce the structural and orientational stability of an α-helical peptide immobilized on the surface but also promote the peptide to
Figure 9. Radial distribution functions, averaged over the last 10 ns of the simulation time in Simulation II, for CF3 carbon (red line), CH2 carbon (green line), and oxygen (blue line) of TFE toward the CH3 carbon of (top) Ala, (middle) Ile, and (bottom) Leu hydrophobic (nonpolar) residues in the CP1 peptide. G
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eventually form a linear α-helical structure standing upright on the SAM surface. By analyzing the local TFE concentration around each amino acid residue, we found that almost all the peptide residues were closely surrounded by the TFE molecules and the TFE molecules aggregate more favorably around the nonpolar (hydrophobic) residues of the peptide. Almost each amino acid residue has a preference for contacting TFE (instead of water), particularly for the amino acid residues containing a nonpolar side chain. Furthermore, with the help of the RDF profiles, we further examined the interactions between the solution (water and TFE) and the peptide, confirming that the preferred aggregation of TFE is mainly caused by its hydrophobic interaction with the peptide. Additionally, this result also suggests that a more organized structure and orientation of TFE molecules around the peptide promotes the TFE preferential surrounding around the peptide. In conclusion, our simulation results can be well correlated to the corresponding SFG experimental observations. The study also further revealed that the TFE aggregation around the surface immobilized peptide provides a local hydrophobic microenvironment that contributes to reduce the destabilizing effect on the structure and orientation of the surfaceimmobilized peptide caused by the hydrophobic interactions between the peptide nonpolar amino acids and water. The TFE aggregation also promotes the immobilized peptide to eventually form a linear α-helical structure with an upright orientation on the surface. Such information provides in-depth atomic-level understanding on the effect of chemical environment (e.g., water or TFE/water cosolvent) on the conformational/orientational behavior of biological molecules immobilized on surfaces.
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AUTHOR INFORMATION
Corresponding Authors
*N. He. E-mail:
[email protected]. Phone: 86-25-83790885. *Z. Chen. E-mail:
[email protected]. Phone: 1-734-615-4189. *C. L. Brooks. E-mail:
[email protected]. Phone: 1-734647-6682. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Key Program for Developing Basic Research of China (2010CB933903 and 2014CB744501), the National Natural Science Foundation of China (61271056 and 21303015), and U.S. Army Research Office (W911NF-11-1-0251). We also acknowledge Shanghai Supercomputer Center for providing computing resources.
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