Enhanced Understanding of Amphipathic Peptide Adsorbed Structure

Oct 31, 2013 - Michael A. Donovan , Yeneneh Y. Yimer , Jim Pfaendtner , Ellen H. G. Backus , Mischa Bonn , and Tobias Weidner. Journal of the American...
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Enhanced Understanding of Amphipathic Peptide Adsorbed Structure by Modeling of the Nonlinear Vibrational Response Sandra Roy, Tsuki L. Naka, and Dennis K. Hore* Department of Chemistry, University of Victoria, Victoria, British Columbia, V8W 3V6, Canada ABSTRACT: We have used molecular dynamics simulations to study the structure and nonlinear vibrational spectra of a face-wise amphipathic α-helical peptide when adsorbed onto hydrophobic, neutral, and charged hydrophilic surfaces from the solution state. Visible-infrared sum-frequency spectroscopy is a powerful probe of interfacial structure, and is unmatched in its sensitivity and specificity for molecules at the buried solid− liquid interface. However, although the resulting experimental spectra serve as fingerprints for the adsorbed-state structure, there is currently limited understanding in how to extract quantitative structural information. We compare our structural results and simulated spectroscopic response with the results of several experimental studies in the literature. We are able to reproduce all of the main features in the experimental observations, and thereby provide some additional fundamental insight into the interaction of this important class of molecules with surfaces of varying hydrophobicity and charge.

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stable α-helix with Lys side chains directed out from one face of the helix, and Leu side chains out from the other. There have been many studies of this molecule adsorbing to a wide variety of solid surfaces over the past three decades, utilizing techniques including electronic circular dichroism (CD),17,18 IR absorption,,17 quartz crystal microbalance (QCM),18,19 Xray photoelectron spectroscopy (XPS),20 time-of-flight secondary ion mass spectrometry (ToF-SIMS),20 near edge X-ray absorption fine structure (NEXAFS),21,22 solid-state NMR,23 and molecular simulations.24,25 Simulations are especially attractive owing to the level of molecular detail they can provide, and their ability to control and tune surface interactions. One of the frontier research efforts in this field is the development of tailored force fields that more accurately describe the interfacial environment, and the transition from the bulk phase to the surface.26−28 On the experimental front, nonlinear vibrational spectroscopy, particularly visible-infrared sum-frequency generation (SFG) spectroscopy, is growing in popularity as a result of its exquisite specificity to the interfacial region, and structural sensitivity. Under the electric dipole approximation, all elements of the nonlinear susceptibility χ(2) vanish in any material with centrosymmetry. For this reason, any peptides in solution will not contribute to the measured SFG response, regardless of the depth of penetration of the probe beams into the solution phase. Molecules immobilized, or merely orientationally constrained, as a result of their interaction with the surface, break the inversion symmetry of the isotropic solution phase, thereby creating χ(2) ≠ 0 and generating an SFG spectrum. By considering multiple energies of the infrared beam throughout the mid-infrared region,

INTRODUCTION The interaction of proteins and peptides with surfaces is of fundamental and practical interest, as it accounts for interactions with membranes, dictates the biocompatibility of implant materials, and is the basis of many biofiltration technologies.1−3 The change in secondary and tertiary structure resulting from the redefined balance between residue−residue and residue−solvent interactions in light of the new residue− surface interactions is the central topic in these application areas.4−7 Face-wise amphipathetic peptides are an important class of natural and synthetic molecules whose structure in solution consists of a rigid core (often an α helix), with hydrophobic residues predominantly on one side, and hydrophilic residues on the other. Such molecules have widespread use as antimicrobial agents.8−11 One theory as to their action is termed the carpet model, where they destabilize bacterial cell membranes by adhering to their surface.12 Another mechanistic theory, termed the barrel stave model,13 is that they insert themselves into membranes, forming a pore with neighboring peptides as a result of their hydrophobic interaction with the membrane lipid tails. The pore with hydrophilic interior then serves as a channel by which contents of the cytoplasm may be exchanged with the extracellular matrix, thereby destroying the cells. In addition to such antimicrobial action, face-wise amphipathetic peptides play an important role in pharmacology, due to their ability to penetrate the cell membranes for drug delivery.14−16 Finally, these molecules, owing to their structural stability and regularity, provide valuable model systems for understanding hydrophobic and charge interactions between biological systems and synthetic materials. One of the most well studied peptides in this class is a 14mer consisting of leucine (L, Leu) and lysine (K, Lys) residues, Ac-LKKLLKLLKKLLKL-COO−, first synthesized by DeGrado and Lear.17 Named LKα14, in solution this molecule forms a © 2013 American Chemical Society

Received: September 16, 2013 Revised: October 28, 2013 Published: October 31, 2013 24955

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surfaces.18,19,21−23,47,48,57 In the last part of this paper, we compare our results with those experimental observations. We will illustrate that we have been able to reproduce all of the gross features in the experimental SFG spectra, and many of the subtleties. This provides valuable insight into the origins of the spectral response, furthering the utility of SFG spectroscopy for the quantitative analysis of biomolecules.

various chemical function groups of the adsorbed peptide may be brought into resonance, thereby providing a fingerprint of the adsorbed structure in the vibrational response. However, despite the significant advancements in instrumentation, techniques, and accompanying analyses, deducing the structure of the molecules responsible for generating the SFG spectrum remains a challenge, even for small molecules. Much effort has been devoted to this pursuit, owing to the paucity of techniques with sufficient selectivity for the adsorbed state. Although there have been detailed SFG studies of individual amino acid interactions with various solid surfaces,29−35 and an increasing number of protein adsorption experiments monitored by SFG,36−46 there have been considerably fewer SFG studies of peptides.18,19,21−23,47,48 There are two primary challenges associated with structural elucidation of large molecules using SFG spectroscopy. One is associated with the large degree of structural heterogeneity in the conformations and orientations of the adsorbed state. We have recently demonstrated that molecular dynamics simulations may aid in such cases, as a numerical ensemble may be generated to create the lab-frame average response that may be considerably more complicated than what may be achieved using an analytical form of an orientation distribution function.32 Our approach also illustrated that multiple conformers may be identified in such simulations, and then used as a basis for modeling the electronic structure that ultimately gives rise to the spectroscopic response. The second challenge is that, for large molecules, the hyperpolarizability of all normal modes may be difficult or impossible to calculate. It has recently been established that techniques that divide the response up into fragments, such the response of each residue, may be successfully applied to generate the optical properties of large molecules such as proteins.49−53 Overcoming these obstacles enables simulation of the experimental SFG response, thereby providing a deeper level of structural understanding.54−56 To date, these techniques have focused on protein and peptide backbone modes, such as those comprising the amide I band. Understandably, these vibrational signatures provide a means to directly elucidate the secondary structure as is performed with linear IR absorption or Raman spectroscopy, or vibrational circular dichroism (VCD) or Raman optical activity (ROA) measurements. However, for molecules such as surfacebound face-wise amphipathic peptides, there is much information available from studying the side chain interactions. In this work, we combine all of these emerging analysis modalities to study LKα14 adsorbed adsorbed onto hydrophobic, neutral-, and charged hydrophilic surfaces. In the next section, we will first provide details of our simulations, including a description of the model substrates and their properties. We will describe the procedure by which we generate the SFG response for the peptide on a residue-byresidue basis, taking its orientation on the surface and conformation into account. We then discuss our findings, first in terms of the backbone and side chain structure of LKα14 adsorbed onto the three surfaces. Finally we present and discuss our model SFG spectra for the molecule as a whole, and also broken down into spectral contributions from only the hydrophilic or hydrophobic components. We also present the SFG spectra on a per residue basis for a deeper insight into the connection between the side chain structure, surface interactions, and measured response. There have been many experimental SFG studies of LKα14 adsorbed onto solid

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METHODS Molecular Dynamics Simulation Details. Surfaces with different wetting behavior have been created. The substrate was intended to mimic an organic self-assembled monolayer (SAM), and consisted of straight chains of OPLS/AA58 methylene united atoms assembled in a hexagonal fashion, with lattice constants a = 4.38 Å and c = 1.60 Å as in ref 59. In the case of the hydrophobic surface (12 centers spanning 17.6 Å), the terminal atom was an OPLS/AA methyl united atom. For the neutral hydrophilic surface, hydroxyl termination was added to 10-atom chains, resulting in an overall length of 18.2 Å. The OH groups were oriented 71.5° from the surface normal and with a randomized azimuth about the normal. A previous Monte Carlo study by Janeček et al.59 investigated the effect of the azimuthal angle of surface OH groups on the hydrophobicity of the surface and determined that randomly distributed and azimuthally frozen OH groups resulted in the same water contact angle as freely rotating OH groups. We have therefore fixed the azimuth of the surface OH groups after their initial randomly generated orientation. For the charged hydrophilic surface, carboxyl and carboxylate termination were added. Since a carboxylated SAM has approximately 50% of its surface charged at pH 7.4,60 we created a 1:1 ratio between carboxyl and carboxylate termination. As a random placement on such a small scale tends to create unwanted agglomeration, we picked every second chain for a charged termination. For the sake of consistency, we used a randomized azimuth about the normal, and fixed the surface coordinates through the simulations. Na+ ions were added to get a charge-neutral system. The amphiphatic Leu-Lys α-helical peptide LKα14 (AcLKKLLKLLKKLLKL-COO−) was modeled with an OPLS/AA force field.58 We have selected OPLS/AA as it has been shown to be compatible with SPC/E water.61 This is important for us as we wish to consider the surfaces in the same state in which they have been characterized, and compared to experiments, in our previous study.62 The peptide was initially created as a perfect α helix, with backbone torsional angles ϕ = −57° and ψ = −47°, an acetylated N terminus, and a charged C terminus. As we model a pH of ≈7, Lys side chains were terminated with NH+3 . To obtain a charge-neutral system, the peptide was accompanied with 5 Cl− ions when in the bulk or in presence of a neutral surface. In the case of the charged hydrophilic surface, 5 Na+ counterions were removed when the peptide was inserted into the simulation cell. The system was solvated with approximately 6500 SPC/E water molecules, depending on the precise area of the specific surface. Temperature was maintained at 300 K with the aid of a Berendsen thermostat.63 Simulations were carried out with 3D periodic boundary conditions. Electrostatic interactions were handled by particle mesh Ewald summation with a real-space equivalent cutoff of 12 Å; van der Waals interactions were cutoff at 12 Å. An initial 1 ns simulation was made with the peptide fixed in space so that the water could first equilibrate with the surface. For the hydrophobic and neutral hydrophilic 24956

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Figure 1. Snapshots of the simulations showing LKα14 in proximity with the (a) hydrophobic, (b) neutral hydrophilic and (c) charged hydrophilic surface. Water molecules have been removed for clarity.

surfaces, simulations were carried out for 50 ns, sampling positions each 100 fs. For the charged hydrophilic surface, the trajectories were obtained over 100 ns as it took longer for the peptide to land on the surface. In all cases, all analyses were performed by averaging over a minimum of 10 trajectories. Figure 1 show snapshots of the three different systems when the peptide is near the surface. Water Contact Angles. Contact angle simulations have been performed to characterize the degree of hydrophobicity and hydrophilicity of the solid substrates prepared. An 895molecule droplet of water was placed on a 156 × 145 Å2 surface. The same equilibration and MD simulation as described above was performed, but for a period of 1 ns. The change in the droplet shape was correlated to its contact angle θc according to the procedure described in ref 64. This resulted in a contact angle of θc = 30 ± 3° for the hydrophilic surface, in agreement with results of other simulations.65 A contact angle of 155 ± 3° was obtained for the hydrophobic surface, also consistent with what has been observed for other solid hydrophobic surfaces.66,67 As the droplet spreads more on hydrophilic surfaces, very large areas are required for these simulations so that the water does not reach the edge of the periodic box. We were not able to determine the contact angle for our charged hydrophilic surface by this method, but have observed that it is considerably smaller than that of the neutral hydrophilic surface (θc < 3°). It is reasonable that it approaches 0° for this type of surface.68,69 Nonlinear Vibrational Spectra. To compute the visibleinfrared sum-frequency spectra for LKα14 adsorbed at the solid surfaces, we consider only the contribution of the residue side chains to the observed response in the region 2800−3400 cm−1. This covers the C−H and N−H stretching modes monitored in experimental studies of the side chains. The SFG intensity is proportional to the magnitude squared of the effective secondorder susceptibility χ(2) eff , and the incident visible (Ivis) and infrared (IIR) laser beam intensities. Elements of χ(2) eff are related to the χ(2) tensor elements via the local field corrections and unit polarization vectors.70,71 As a prerequisite step, the hyperpolarizability α(2) is required for every vibrational mode of each Leu and Lys side chain. However, this electronic property is sensitive to the conformation of the chains.32 It is therefore necessary to first identify which side chain conformers are present when the peptide is on the surface. Populations of the 5 relevant dihedrals of the Lys side chain are shown in Figure 2a, revealing 3 populations. Those for the two Leu dihedrals are shown in Figure 2b, also indicating 3 populations. A correlation analysis

Figure 2. Distribution of (a) Leu side chain dihedral angles L1 and L2, and (b) Lys side chain dihedrals K1−K5.

within the dihedrals of the same amino acid was performed to find the principle conformation of the side chains. Proceeding only with the major populations, we were left with 3 Leu conformers and 7 Lys conformers. Using the isolated Leu and Lys amino acids as representations of the Leu and Lys residues, initial geometries of these 10 side chain conformers were prepared, and then optimized via B3LYP/6-31G(d) using GAMESS with a PCM solvent model while constraining the dihedrals to the desired values for the conformers of interest. As the Hessian needs to be evaluated at a saddle point on the potential energy surface, in a subsequent step, all constraints were lifted, and the optimization was repeated. Investigation of the resulting structures revealed that the dihedral angles of interest did not differ from their starting angles by more than 2°. The final stage is to calculate the contribution to hyperpolarizability from each normal mode ων > 2000 cm−1 for each of the 10 structures. To account for the fact that we are approximating residue side chain modes with isolated amino acid side chain modes, the backbone terminal NH+3 symmetric and antisymmetric stretching vibrations were excluded. In the case where the visible beam is far from resonance, we can write the l,m,n element of the molecular second order polarizability, α(2), as (2) αlmn , ν(ω IR ) =

̂ |e⟩⟨e|μn̂ |g ⟩ ⟨g |αlm ων − ωIR − i Γν

(1)

where |g⟩ is the vibrational ground state wave function, |e⟩ is the vibrational first excited state wave function, α̂ is the transition polarizability operator, μ̂ is the transition dipole moment operator, ων is the normal-mode frequency, i = √−1, and Γν is 24957

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the homogeneous line width. The indices l,m,n represent any of the molecular-frame Cartesian coordinates a,b,c. All 27 elements of α(2) are calculated following the scheme we have described previously.31,32,72,73 Briefly, we make the harmonic approximation (2) αlmn , ν(ω IR ) =

(1) 1 ∂αlm ∂μn 1 2mν ων ∂Q ∂Q ων − ωIR − i Γν

(2)

where mν is the reduced mass of each normal mode and Q is the normal mode coordinate. These two quantities are determined from a Hessian calculation. Here α(1) is the linear polarizability, and μ is the dipole moment; their derivatives with respect to Q are calculated numerically with B3LYP/G-31(d) and PCM solvent as described previously.31,32,72,73 The complete hyperpolarizability at the IR probe beam frequency ωIR is given by (2) αlmn (ωIR ) =

(2) ∑ αlmn , ν(ω IR )

(3)

ν

We can ignore any nonresonant contribution to α as we will later consider only the imaginary component of the SFG spectra. The hyperpolarizability in the surface frame α(2) ijk is obtained by projection, where i,j,k are any of the laboratory (surface) frame Cartesian x,y,z components.74 This is accomplished with the direction cosine matrix D (2)

⎡(x·̂ a)̂ (x·̂ b)̂ (x·̂ c )̂ ⎤ ⎥ ⎢ D = ⎢(y ̂ ·a)̂ (y ̂ ·b)̂ (y ̂ ·c )̂ ⎥ ⎥ ⎢ ⎥ ⎢ ̂ ⎣(z ·̂ a)̂ (z ·̂ b) (z ·̂ c )̂ ⎦

(4) Figure 3. (a) LKα14 end-to-end distance, dee, (b) distance between the LKα14 center of geometry, dCOG, and the surface, (c) long axis tilt angle, θP. Results for the peptide in bulk solution (when not adsorbed on any surface) are indicated in black. In the case of bulk solution, dCOG is calculated with respect to an arbitrary point in the simulation cell. Results of the hydrophobic surface are shown in blue; neutral hydrophilic surface in red; charged hydrophilic surface in green.

to write abc abc abc (2) αijk =

(2) ∑ ∑ ∑ DilDjmDknαlmn l

m

n

(5)

χ(2) ijk

The second-order susceptibility is then the straightforward summation of lab-frame hyperpolarizabilities χijk(2) =

1 ε0

∑ αijk(2) N

distribution full width of 4 Å. On the neutral hydrophilic surface, the distribution is slightly broader and closer to the interface. On the charged hydrophilic surface, the peptide center of geometry is the closest to the surface with a relatively narrow distribution. As expected, an isotropic distribution of dCOG (with respect to the simulation box origin at z = 0) is observed in the bulk. The end-to-end distance, dee was defined as the distance between the N- and C-terminal carbon atoms. These results are shown in Figure 3b for LKα14 adsorbed on the surfaces and in the bulk. For comparison, LKα14 in a perfect α-helical conformation (all backbone torsional angles set to ϕ = −57° and ψ = −47°) has an overall length of 21 Å, as indicated by the vertical dashed line. However, in solution we observe two populations. One has dee near the ideal α-helix value; the other, dominant, population is centered near 12 Å. At the hydrophobic surface (blue trace), the two populations are much more separated, and neither corresponds to the dee of an ideal α-helix. One population is found at 10 Å, and the other at 23 Å. The neutral hydrophilic surface has a peak at short distance (10 Å) and a shoulder near the ideal helical dee. The charged hydrophilic surface displays a very broad peak centered at the ideal α-helix distance of 21 Å.

(6)

where N is the number of frames over the course of the MD trajectory, with one LKα14 molecule per frame. Only frames when LKα14 is adsorbed were considered in the above summation.

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RESULTS End-to-End Distance, Center of Geometry, Long Axis Tilt Angle. We investigated the overall peptide characteristics before analyzing the side chain and backbone conformations in detail. We first determined dCOG as the distance between the peptide center of geometry and the surface. Based on results obtained for 10−15 trajectories for each surface, we arrived at cutoff values for the adsorbed states. LKα14 was considered to be on the surface if its center of geometry was less than 15 Å (from either surface in the periodic cell). In Figure 3a we display the dCOG distribution when adsorbed on the hydrophobic surface (blue), neutral hydrophilic surface (red), and charged surface (green). For comparison, results obtained for the bulk water simulations (no surface present) are shown in black. In the case of the hydrophobic surface, we notice that adsorption occurs around 13 Å from the surface, with a 24958

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Figure 4. Ramachandran plots illustrating ψ versus ϕ backbone torsional angles of LKα14 in (a) bulk water, and adsorbed at (b) a hydrophobic surface, (c) neutral hydrophilic surface, and (d) charged hydrophilic surface. Higher populations appear darker (black). The boundaries of the rightand left-handed α-helical and β sheet regions are according to Morris et al.75 Core right-handed α-helical regions are labeled A, allowed regions a, generous regions -a. Core left-handed α-helical regions are labeled L, allowed regions l, generous regions -l. Core β-sheet regions are labeled B, allowed regions b, generous regions -b.

random coil structures as has been previously identified.24 When the peptide is adsorbed at the hydrophobic surface, Figure 4b shows two additional minor populations. One appears at (ϕ ≈ −110°, ψ ≈ −130°), the other is at (ϕ ≈ −150°, ψ ≈ −150°). These same four populations are observed at the neutral hydrophilic surface, noting that the α-helix is not as predominant here. When excluding the initial adsorption events from our analysis, the α-helix population increases (along with the random coil), but the populations at positive ϕ disappear. For the charged surface, populations are present also in both the α-helical region and the β-sheet regions. The populations at positive ϕ are not present, but we do notice a very small population around (ϕ ≈ −90°, ψ ≈ −0°). There is a general trend that the α-helical content decreases in favor of the (ϕ ≈ −80°, ψ ≈ −150°) population from the hydrophobic surface to the charged hydrophilic surface, as has been observed in previous studies using the same forcefield.24,25 Side Chain Orientations. In order to more directly study the effects of the surface on the helix structure, we have monitored the tilt of the individual side chain long axis vectors with respect to the surface normal. In the case of Leu, this vector points from the backbone Cα to the isobutyl central carbon atom. An orientation of 0° would indicate that the isobutyl group is pointing away from the surface. An orientation

Finally, we generate histograms of the helix long axis tilt angle, θP, with respect to the surface normal (or with respect to the box z vector in the case of the bulk simulation). This long axis vector was defined using the same atoms as for dee, pointing from the N to the C terminus. In all cases, these histograms were normalized with respect to an isotropic distribution (dividing by sin θP). As expected, the bulk water θP distribution (black trace in Figure 3c) is flat, indicating free tumbling in solution and no orientation preference. At the hydrophobic surface (blue trace), LKα14 is adsorbed with a relatively narrow range of tilt angles centered at 75°. The peak of the tilt distribution occurs at 80° (long axis nearly parallel to the surface), and falls sharply toward larger tilt angles. The tail of the distribution is almost solely toward smaller (steeper) angles. Wider distributions are seen for the neutral hydrophilic surface, centered at 60°. A strong orientation preference centered near 90° is present for the charged hydrophilic surface with a tail toward higher angles. Backbone Conformations. Ramachandran plots showing peptide backbone ϕ and ψ torsional angles are shown in Figure 4. For the bulk water simulations, Figure 4a reveals a dominant population in the middle of the classical core α-helical region. A secondary minor population is evident around (ϕ ≈ −80°, ψ ≈ −150°), in the classical β-sheet region, but originating from 24959

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Figure 5. Side chain orientations determined by the tilt θS of the longest axis that connects the main chain to the side chain. The inset (lower right) shows that this vector points from the α-carbon atom to the isobutyl methine carbon in the case of the Leu residues, and from the α carbon to the Lys terminal N atom. Residues are labeled starting at the N terminus. Results obtained when LKα14 is adsorbed on the hydrophobic surface are shown in blue; on the neutral hydrophilic surface in red; on the charged hydrophilic surface in green. The bulk solution results are shown in black for comparison.

Figure 6. Side chain orientations determined by the tilt θT of the chain end vectors. The inset (lower right) shows that for Leu residues, the vector starts at the isobutyl methine carbon and bisects the methyl groups. For Lys, we have chosen the NH+3 3-fold symmetry axis. Residues are labeled starting at the N terminus. Results obtained when LKα14 is adsorbed on the hydrophobic surface are shown in blue; on the neutral hydrophilic surface in red; on the charged hydrophilic surface in green. The bulk solution results are shown in black for comparison.

of 180° would indicate that it is pointing toward the surface (for example, on the face of the helix in contact with the surface). For the Lys residues, this vector points from the backbone Cα to the side chain ammonium N atom. Likewise an orientation of 180° would indicate that the lysine side chain terminal is directed toward the surface. The histograms shown in Figure 5 follow the same color convention as we have used

previously. In the bulk solution, the black traces indicate an isotropic distribution of side chains as the peptide is tumbling. At the hydrophobic surface (blue traces), we generally observe a strong tendency for LKα14 to adsorb such that the hydrophobic Leu residues are directed toward the surface. In particular, about half the Leu residues (Leu5, Leu12, Leu14) have narrow orientation distributions peaked at 180°. The 24960

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(θT > 90° in Figure 6). A similar observation was made of the Leu residues, with Leu12 and Leu14 overall side chains directed

remaining Leu residues are still directed toward the surface but slightly tilted, peaked around 120°. The orientation distributions of the Lys residues differ more according to their position along the chain. No Lys residue has a side chain directed toward the surface, but some (Lys3, Lys6, Lys9) display a population laying parallel to the surface (close to 90°). In the case of the neutral hydrophilic surface at short times (solid red traces), the Leu residues display very little preference in their orientation with respect to the surface. The orientation distributions are slightly weighted to θS > 140° (toward the surface). Most Lys residues (Lys2, Lys3, Lys9, Lys10) have two distinct populations. The dominant one is peaked at 0°; surprisingly, this directs the hydrophilic side chains away from the hydrophilic surface. A second population for these four residues is observed near 120°. On the charged surface, most Lys show a preference of tilt angle θS > 90°, with the exception of Lys9 and Lys6. For the leucine, the tilt angle varies with the placement in the chain, although the trend seems to be more toward 90°, avoiding the extremes (0°, 180°). In the following section, we will describe the results of our simulated SFG spectra. In the case of vibrational spectroscopy experiments such as SFG, it is common to characterize the side chain orientations based on the observed functional group resonances. In particular, the CH3 and NH+3 symmetric and antisymmetric stretching bands serve as markers for the side chain orientations. However, as there is some flexibility in the side chain conformations, we must keep in mind that these resonances are indicators of the ends of the side chains, rather than the average direction of the extended side chain. For this reason, we have performed a second side chain analysis, based on the vector that bisects the two terminal methyl groups of the Leu isobutyl chain, and the 3-fold symmetry axis of the Lys NH+3 group. These atoms are often isotopically labeled for easy identification in the vibrational spectrum. We make the connection to this spectroscopy by defining these shorter side chain ‘end’ vectors that correspond more closely to the probed functional groups. In the case of Leu, we defined this vector from the isobutyl central carbon atom to the midpoint between the two methyl carbon atoms. For Lys, the vector runs from the ammonium N atom to the midpoint of the three hydrogen atoms. These terminal group distributions, termed θT, are shown in Figure 6. In the hydrophobic case, the short vectors of the Lys side chains are not lying in the plane of the surface. These vectors (Lys3, Lys6, Lys9) are observed to be pointing away from the surface (θT < 45). Lys3 also has a second population centered at 70°. For the charged surface, the trend starts to be more visible when analyzing the short vectors. The Leu have mostly a population around 90° while the Lys clearly have an orientation distribution peaked around 60−70°. Major features in this chain-end analysis are mostly the same as observed in the case of the longer side chain vectors, especially for the neutral hydrophilic surface. The bulk water results, and the distributions for some of the residues on the surfaces, are observed to be more evenly distributed between 0−180°. This is reasonable, considering that shorter vectors are more orientationally averaged in a given time. In other words, even when the overall side chain is oriented, some additional flexibility remains in the chain end. However, there are some notable exceptions in the results we observe for the charged hydrophilic surface (green traces). In the case of Lys6 and Lys9, there is an obvious change in direction. While the long axis of the side chain is pointing toward the surface (θS > 90° in Figure 5), the terminal NH+3 axes are pointing away from the surface

Figure 7. (a) Imaginary χ(2) ssp spectra of LKα14 plotted with a broken wavenumber axis to highlight the aliphatic C−H stretching region between 2800 and 3000 cm−1 and the NH+3 stretching region between 3200 and 3400 cm−1. The response has also been separated into (b) contributions of only the Leu residues and (c) contributions from only the Lys residues. Results obtained when LKα14 is adsorbed on the hydrophobic surface are shown in blue; on the neutral hydrophilic surface in red; on the charged hydrophilic surface in green.

slightly toward the charged hydrophilic surface (not much preference), but with the methyl groups clearly pointing away from the surface. Nonlinear Vibrational Spectra. Vibrational SFG spectra were computed by first analyzing the structure of each peptide residue side chain for every snapshot in the simulation. A scoring function was used to determine which Leu or Lys conformer best described the particular side chain, and the corresponding values of α(2) for the specific modes of interest were then used as described in the Methods section. Once appropriate hyperpolarizability values for each of the 14 residues were identified, a projection into the surface-fixed frame resulted in the nonlinear susceptibility tensor χ(2). As we seek comparison with results of experimental studies performed probing s-polarized SFG using s-polarized visible and ppolarized infrared beams (the so-called ssp scheme), we (2) (2) calculate χ(2) ssp ≡ 1/2 (χxxz = χyyz). These elements are equivalent in the case of azimuthal symmetry, and we benefit from improved statistics by averaging them in our simulations. The overall imaginary component of the SFG line shape, as would be obtained in a heterodyne SFG experiment, is plotted in Figure 7a. The sign of the imaginary component may be related to the direction in which the chemical group is pointing. Such analysis is particularly applible to the CH3 and NH+3 symmetric stretching modes, as the orientation of the hyperpolarizability with respect to the local mode C3 axis is evident. This is the reason why, in addition to orientational analysis of the side chain long/main axis vectors (Figure 5), we have additionally studied the end group orientations (Figure 6). In our case, when the Leu isobutyl group are directed away 24961

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Figure 8. Simulated χ(2) ssp spectra for every residue, labeled starting at the N terminus. Results obtained when LKα14 is adsorbed on the hydrophobic surface are shown in blue; on the neutral hydrophilic surface in red; on the charged hydrophilic surface in green. The scale for each of the Leu (2) residues is the same, with −0.25 ≤ Im[χ(2) ssp ] ≤ 0.25. Similarly, the Lys spectra all have the scale of −0.05 ≤ Im[χssp ] ≤ 0.05.

from the solid surface, or when the Lys NH+3 group is directed away from the solid surface, Im[χ(2) ssp ] > 0. When we observe Im[χ(2) ] < 0 for either of these bands, we know that the ssp respective functional groups are pointing toward the surface. Note that this convention is opposite in sign to what is commonly used for phase-resolved SFG measurements at solid−air and air−water interfaces,73,76,77 as a result of the opposing definition of the surface normal. One of the advantages of spectral simulations is that targeted contributions may be isolated for analysis, similar to what can be obtained experimentally by isotopic substitution. For example, we can break down the response into separate contributions from the Leu and Lys residues. In the Leu SFG spectra (see Figure 7b), the most prominent peak around 2870 cm−1 corresponds to the CH3 symmetric stretch. The small peaks near 2900 cm −1 correspond to CH 3 and CH 2 antisymmetric stretching. The CH2 symmetric mode is present just below 2850 cm−1. In the Lys SFG spectra (see Figure 7c), the dominant peak around 2860 cm−1 originates from CH2 symmetric stretching, as well as a smaller antisymmetric contribution at 2875 cm−1. The bands between above 2900 cm−1 correspond to the different CH2 antisymmetric stretches. The NH3 symmetric and antisymmetric stretches are present around 3250 cm−1 and 3340 cm−1, respectively. It is to be noted that an isotope labeling study of the lysine N atoms for LKα14 on hydrophobic and hydrophilic surfaces57 has shown that the signal observed in the 3200−3400 cm−1 region may be attributed solely to the NH+3 from the side chains, with no contribution from the backbone N−H stretching. Similarly, our model considers only the side chain vibrational modes. Strong SFG signal in the CH region (2800−3000 cm−1) can be observed for all three surfaces in the overall spectra. Though as we look at the contribution the hydrophobic and hydrophilic components separately, we can notice that most signal for the charged surface comes from the lysine residues while for the hydrophobic surface, it almost uniquely arise from the Leu contribution. The relative intensity of these modes provides a qualitative description that Leu residues are strongly ordered when LKα14 is adsorbed on the hydrophobic surface. The χ(2) response on the neutral hydrophilic surface (blue traces in

Figure 7 is quite different. We notice that the N−H stretching becomes slightly stronger, and the methyl stretching is weaker. As an additional bonus, our approach enables us to isolate the contribution of the individual residues in the peptide. This has been done for the imaginary component of χ(2) ssp as shown in Figure 8. For the Leu resides, we show the aliphatic C−H stretching region from 2800−3000 cm−1; for the Lys residues we show the N−H stretching region froom 3200−3400 cm−1. For the hydrophobic surface, the Leu residues all contribute to a negative band in the C−H stretching region, with the exception of Leu11, which displays positive CH2 and CH3 antisymmetric peaks, and Leu14, which shows a positive feature for its CH2 symmetric stretch. On the neutral hydrophilic surface, the Leu contribution to the spectra is similar throughout the peptide, with positive bands, except for a small negative contribution from Leu11 antisymmetric modes. There is almost no contribution from Leu residues to the SFG response on the charged hydrophilic surface, with small positive and negative features distributed among the residues. For the Lys residues, there is no strong trend evident in the contribution of the NH+3 stretching to the hydrophobic surface SFG response. However, on the neutral hydrophilic surface, there is a clear tendency for the Lys residues to contribute negative bands to the N−H stretching region, with the exception of Lys3 and (to a smaller extent) Lys6. On the charged surface, the Lys contribution is consistent with a positive contribution to the NH+3 symmetric stretch.

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DISCUSSION Hydrophobic Surface. In the center of geometry analysis (Figure 3a), we observed that LKα14 adsorbs on the hydrophobic surface at a distance of ≈11 Å from the surface. The end-to-end distance (Figure 3b) shows a strong peak at 10 Å and a smaller one at 23 Å, meaning that one structure corresponds to an almost-perfect α helix. This is further evidenced by the strong feature at (ϕ ≈ −70°, ψ ≈ −40°) in the Ramachandran plot (Figure 4b). Since the Ramachandran plot is dominated by α helical features, and the largest population end-to-end distance is 10 Å, we can infer that some of these peptides are α helices containing a fold, kink, or part of a random coil. Analyses of the amide I region in experimental

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SFG signal21 have led to the same conclusion, where LKα14 adopt predominantly α-helical conformations when adsorbed on a hydrophobic (dodecanethiol) SAM surface. The same conclusion was obtained from molecular dynamics performed by the Latour group.24 The long axis tilt angle peak distribution being tailed mostly on the θS < 90° side means that even if most of LKα14 are lying down on the surface, the N terminus is preferentially adhering to the surface. This is the side where the molecule lands on the surface. This is logical since the acetylated end is less hydrophilic than the carboxyl C terminus. Also, the acetylated group is pointing on the same side as the hydrophobic residues side chains in the perfect α helix. Side chain tilt angles (Figures 5 and 6) show that leucine side chains are pointing toward the interface, optimizing the nonpolar interactions of the methyl from the amino acids and surface. This interaction was demonstrated in an experiment by Apte et al. where they investigated LKα14 adsorption on SAM surfaces with methyl termination by means of XPS and ToF-SIMS experiments.20 With a perfect α helix present, most of the lysine residues are pointing on one side of the peptide, and most of the leucine are pointing on the other side. Since LKα14 shows an α helix feature when adsorbed at the hydrophobic interface, it is normal to see the lysine counterpart having their residues pointing mostly away from the surface with θS < 90°. This can be seen for the lysine residues in the long vector tilt angle distribution (Figure 5) showing broad values of tilt angle is seen that can be correlated to movement in the long side chain as there are no surface interaction to hinder its movement. The main peak in the SFG spectra (Figure 7a) is present around 2870 cm−1 and is from the Leu contribution (see Figure 7b). This peak corresponds to CH3 symmetric stretch and arise from a strong ordering of the leucine toward the surface, which is in agreement with our orientation analysis showing most isopropyl directed directly toward the surface (θT = 0°). For the lysine counterpart, while the NH+3 points slightly away from the interface, the tilt at which it is oriented, compared to the leucine, will affect the relative intensity of the SFG signal. We can thus see almost no population at the N−H vibrational modes (≈ 3300 cm−1). Since the lysine side chains are not constricted by the surface, they can move more freely, causing a cancellation in the SFG signal both for the CH2 and NH3 signal. The same behavior was found experimentally by Mermut et al. 19 when studying LKα14 at deuterated polystyrene surface. They found a strong signal at 2869 cm−1 attributed to CH3 symmetric stretching, and no distinct signal in the N−H vibrational region. This agrees with our simulated spectrum quite well as one peak dominates our SFG spectrum. Supporting experimental results can be found from Phillips et al.,18 and from York et al.,47 and similar results from a fluorocarbon surface had been achieved by Weidner et al.22 phase analysis of the CH3 SFG response using a gold underlayer at a hydrophobic SAM surface further supported the notion that the methyl groups are pointing toward the surface.21 This may be directly correlated to our side chain tilt angle results (see Figure 6) indicating a strong θT = 0° preference for the leucine. Weidner et al. additionally used SFG together with solid-state NMR to assess the orientation of LKα14 adsorbed on polystyrene.23 The authors deuterated each leucine side chain separately and obtained SFG spectra corresponding to every hydrophobic position along the peptide. From the NMR data, they concluded that Leu1, Leu4, Leu8, and Leu11 are interacting more with surface. This supports our

results as we observe (Figure 6) that Leu1 and Leu8 have the strongest orientation preference toward the surface. In a study of LKα14 adsorbed to hydrophilic MUDA (mercaptoundecanoc acid) and hydrophobic dodecanethiol surfaces, Weidner et al. concluded, from the SFG response in the amide I region (≈ 1650 cm−1), that the peptide adopts an α-helical conformation in both environments.21 Their results from analysis of the methyl phase relative to the gold substrate led the authors to conclude that the CH3 is pointing toward the surface in the case of the hydrophobic surface. They additionally found that the intensity of the peptide methyl mode was strongest in the case of the hydrophobic surface. This is in agreement with our structural analysis that reveals a stronger orientation of isopropyl oriented toward the surface. This is also observed in the corresponding SFG signal, a negative peak in the Im(χ(2) ssp ) spectra. Neutral Hydrophilic Surface. In the distance analysis (Figure 3a), we observed that LKα14 adsorbs at ≈10 Å from the neutral hydrophilic surface. The results for the long axis tilt angle have a peak below 90°, similarly, the hydrophobic surface results also show a preference of the N-terminus to be toward the surface. In extended simulations (not shown), we observe an increased population of small angles. This indicates that the N terminus of the chain might used as an anchor point while the peptide reorients itself. A strong α-helical feature in the backbone torsional angles, in conjunction with a short end-toend distance, indicates that the peptide will most likely have a kink somewhere in its chain. Side chain orientation distributions (Figures 5 and 6) for LKα14 adsorbed onto hydrophilic surfaces do not display as strong an orientation preference compared to the two other surfaces. We do notice a recurrent feature in Figure 5 for the lysine side chain with peaks at ≈0° and ≈120°. This could be explained by the similarity between the surface hydroxyl and the surrounding water, where the hydrophilic lysine NH+3 hydrogen bonds with water and the surface. SFG spectra at the hydrophilic surface generated for only the Lys side chains (Figure 7c) reveal a negative peak for the NH3 symmetric stretch and a positive CH2 symmetric stretch. Although the orientation of the Lys side chains is not as pronounced as it is when LKα14 is adsorbed on the other two surfaces, it is clear from the SFG results that Lys still has a preferred orientation and makes a contribution to the SFG signal. A strong positive signal in the leucine CH3 symmetric stretch arises from the isobutyl group pointing away from the hydrophilic surface, in contrast to what we observed for the hydrophobic surface. Charged Hydrophilic Surface. Most SFG experiments of LKα14 adsorption onto hydrophilic surfaces have been performed on SiO2 and mercaptoundecanoic acid (MUDA), and these surfaces bear significant surface charge at neutral pH.19−21,78 Our histogram of the overall peptide backbone tilt orientation (Figure 3c) revealed that the peptide is laying nearly perfectly flat on the charged surface, also explaining the shortest distance between the peptide’s center of geometry and the surface (Figure 3a). Looking more closely, the tilt population is centered at values slightly greater than 90° but tails entirely toward high tilt angles. This indicates a stronger interaction of the C-terminus with the charged surface. This might be explained by electrostatic interaction between the negatively charged carboxylate group and the sodium cations that are attracted to the negatively charged substrate. In addition to the clear α-helical content revealed in the 24963

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surprising results, in terms of the specific backbone and side chain orientations in the corresponding adsorbed states. Our formalism is generally applicable to any peptide or protein, and provides a framework that enhances the power and capability of SFG spectroscopy in the elucidation of biomolecular structure.

Ramachandran plot (Figure 4d), there is significant disorder. This is also logical considering the extremely broad distribution of end-to-end distances when on the surface (Figure 3b). This surface-induced deformation may also be explained by the presence of the cations in the interfacial region. Furthermore, these electrostatic interactions encourage closer contact with the backbone, causing both Leu and Lys side chains to lie close to the plane of the surface (as evidenced by θS ≈ 90° for both types of residues in Figure 5). Paying closer attention to the chain ends, Figure 6 reveals a stronger orientation of the lysine terminal NH+3 to be directed slightly away from the surface. This is also in line with a picture of Na+ accumulation at the planar surface. Finally, we rationalize the same behavior in the sign of the imaginary component of χ(2) in the SFG spectra. Our simulated SFG spectra are in nice agreement with experimental studies at charged silica surfaces in that we observe hardly any CH3 signal from Leu groups.19,78 However, we do observe CH2 stretching from the Lys side chains, where the experimental results observe practically no aliphatic C−H stretching. We believe this may be due to the statistical challenge associated with having enough separate starting configurations to remove the orientation and conformation bias upon initially landing on the surface. Once the peptide lands on the charged surface, strong electrostatic interactions tend to keep it there, removing the possibility of reorientation due to desorption-readsorption events. It is for this reason that we averaged the results of 10−15 runs for each system, hoping also to remove any bias associated with the peptide’s initial landing orientation and conformation. Nevertheless, the SFG CH2 response is especially sensitive to gauche defects in the Lys side chain, and any kinks are amplified in the response. When considering a different charged surface, MUDA, experimental SFG studies did observe some Leu methyl signal, and the phase with respect to gold indicated that methyl groups are directed away from the charged surface.21 Although our results indicated that the Leu and Lys side chains are both lying relatively flat on our model charged surface, looking at the Leu isobutyl chain ends specifically (Figure 6) does indicate a slight upward direction of these groups. We note that our modeled spectrum displays two distinct NH3+ bands, while the experiments show a broad response in the region 3000−3400 cm−1.19,78 This may be due to the contribution of the water O−H stretching in the experiment, a slightly distribution of NH+3 environments in the real system, or the possibility of a slight frequency scaling discrepancy in our simulations.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for support of this science with a Discovery Grant. Computers in our group were purchased with startup funds from the University of Victoria. All simulations longer than 50 ns were run on Westgrid clusters. We acknowledge a 2013 Compute Canada Resource Allocation. We thank Belaid Moa (UVic, Compute Canada, Westgrid) and Kuo-Kai Hung for assistance in migrating our tools onto Compute Canada machines.

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REFERENCES

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CONCLUSIONS Molecular dynamics simulations were applied to study the adsorption of the face-wise amphipathic peptide LKα14 from solution onto hydrophobic, neutral hydrophilic, and charged hydrophilic solid surfaces. In addition to the orientation and structural analyses that are readily obtained from the trajectories of the peptide coordinates, we have developed a formalism that enables us to reconstruct the nonlinear response to simulate visible-infared SFG spectra. This is particularly valuable, as it enables a detailed comparison with experimental results from the literature, and also provides further insight into the origins of specific contributions to the spectra. Our results agree with experimental findings that the N−H stretching from Lys side chains is negligible when the peptide is adsorbed onto hydrophobic surfaces, and CH3 stretching from Leu isobutyl groups is not observed when on charged hydrophilic surfaces. We have been able to understand the structural origins of these 24964

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dx.doi.org/10.1021/jp409261m | J. Phys. Chem. C 2013, 117, 24955−24966