The Role of Hydrophobicity in the Stability and pH-Switchability of (RXDX) 4 and Coumarin-RXDX Conjugate β-Sheets
pH-switchable, self-assembling materials are of interest in biological imaging and sensing applications. Here we propose that combining the pH-switchability of RXDX (X=Ala, Val, Leu, Ile, Phe) peptides and the optical properties of coumarin creates an ideal candidate for these materials. This suggestion is tested with a thorough set of all-atom molecular dynamics simulations. We first investigate the dependence of pH-switchabiliy on the identity of the hydrophobic residue, X, in the bare (RXDX) 4 systems. Increasing the hydrophobicity stabilizes the fiber which, in turn, reduces the pH-switchabilty of the system. This behavior is found to be somewhat transferable to systems in which a single hydrophobic residue is replaced with a coumarin containing amino acid. In this case, conjugates with X=Ala are found to be unstable and both pHs while conjugates with X=Val, Leu, Ile and Phe are found to form stable β-sheets at least at neutral pH. The (RFDF) 4 -coumarin conjugate is found to have the largest relative entropy value of 0.884 ± 0.001 between neutral and acidic coumarin ordering distributions. Thus, we posit that coumarin-(RFDF) 4 containing peptide sequences are ideal candidates for pH-sensing bioelectronic materials.
Introduction<!>System Setup<!>Simulation Details<!>Results and Discussion<!>pH-Switchability of RXDX Fibers<!>The Interplay of Hydrophobic and Coulombic Interactions.<!>Order of Coumarin Sidechains within Coumarin-(RXDX) 4 Fibers<!>Conclusion<!>Supporting Information
<p>Materials that are electronically conductive, tunable, and biocompatible are of great interest for bioelectronic applications. Peptides have been combined with a variety of optoelectronic materials for use in photovoltaic cells 1 and other electronic applications 2 to attempt to attain all of these properties. [3][4][5][6][7][8] These materials combine the self-assembly, biocompatibility, and tunable behavior of peptides 9,10 with the optoelectronic properties of a dye molecule. 11 Stacking of highly π-conjugated dye molecules can lead to electronic delocalization in the self-assembled aggregate and provide the electronic property of interest. Thus, it necessary to predict macroscopic structure from molecular details as the optoelectronic properties of the material depend on the assembled structure.</p><p>Peptides are inexpensive, easily synthesized and provide a rich diversity of selfassembled structures, making them ideal candidates for scaffolding optoelectronic materials. 12 To date, peptides have been used to create molecular wires, 13 switches 14 and surfactants 15 among other materials. 12,[16][17][18] Due to the diverse set of chemical building blocks and resulting assembled structures, peptides continue to be an active area of research in bottom-up materials engineering. 19 The amino acid sequence (RADA) n (Arg-Ala-Asp-Ala) has received a lot of attention over the past decade for its self-assembling properties and applications in tissue engineering. 20 (RADA) n is a self-complimentary peptide sequence comprised of alternating oppositely charged hydrophilic residues (Arg and Asp) and neutral hydrophobic residues (Ala) allowing it to form highly organized β-sheet structures leading to nanofiber and hydrogel formation. 12,[20][21][22][23][24][25][26][27][28][29][30][31] Potentially, the most interesting property of (RADA) 4 is the transformation in the self-assembled structure when the pH is changed. 14,29 Many of the proposed applications for small self-assembling peptides require the inclusion of additional functional components. Peptides have been tethered to aliphatic [32][33][34][35][36] and aromatic groups 37,38 to achieve control over assembly and additional functionality. 9 Aromatic amphiphiles are of particular interest here due to their potential use as bioelectronic and biosensing materials. 38 The peptide is used as a solubilizing and scaffolding agent for the aromatic groups which act as both agents of hydrophobic assembly and electron rich or deficient semiconducting materials. A variety of aromatic groups have been linked to peptides include OPV3, 8,39,40 napthalene 41,42 , perylene diimide, [43][44][45][46][47][48][49][50][51] coumarin, 2,52-54 and others. 38 Here we focus on the use of coumarin due to its high quantum yield, extended spectropscopic range, photostability, and general solubility in a variety of solvents. 2,52,54 Furthermore, many coumarin derivatives have been synthesized that could be used to further tune the optical properties of the material. 2,52,54 Coumarins alone suffer a lack of tunability and biocompatability making them less than ideal candidates for bioelectronic properties, but coumarin derived unnatural amino acids have been synthesized for biological imaging applications allowing it to be easily incorporated into a peptide sequence. 2 The relative importance of hydrophobicity and aromaticity in the formation of βsheet fibrils from self-complimentary amphipathic peptide sequences is debated. [55][56][57][58][59] Generally, a hydrophobic threshold must be reached to have fibrilization in these types of systems. [58][59][60] Some of these studies suggest that aromatic interactions are not important for determining fibrilization rates, but do have an impact on the selfassembly of these materials, specifically the morphology of the fibers. 56,57 It is even less clear how all of these factors will affect the self-assembly and pH switchability of a peptide-dye conjugate.</p><p>In this manuscript, we describe results of a systematic computational investigation of the self-assembly and pH switchability of (RXDX) 4 (X = Ala, Val, Leu, Ile, and Phe) and (RXDX) 4 -coumarin peptides. The set of five amino acids chosen for the X position have incremental change in hydrophobicity, based on water-octanol partition coefficients, 61 allowing us to probe the effect of hydrophibicity on the properties of the materials. Phenylalanine was added to this set to study the effect of adding a residue which is capable of π-stacking. Lastly, a single hydrophobic residue in each (RXDX) 4 fiber is mutated to a coumarin derived amino acid to study the stability and pH-switchability of coumarin-RXDX conjugates.</p><!><p>Simulations are performed on five separate systems of (RXDX) 4 with X = Ala (A), Val (V), Leu (L), Ile (I), and Phe (F). β-strands of each (RXDX) 4 sequence are created using the sequence command in tleap within the AmberTools 18 package. 62 Each system contains twenty molecules of the (RXDX) 4 sequence arranged in two β-sheets, forming a fiber that is ten β-strands long as shown in Figure 2. The initial structure for each system is based on spacings between β-strands provided by Cormier et al. 63 The distance between the two β-sheets are varied based on the identity of the nonpolar residue. For X = Ala, Val, Leu, Ile, and Phe the initial distance for (RXDX) 4 fibers are 6.0, 7.0, 9.0, 9.0, and 11.0 Å, respectively. Each fiber is solvated with TIP3P water and 0.1 M NaCl plus neutralizing ions in a 143 Å cubic box with periodic boundary conditions. To determine the effect of pH on the stability of the fibers, we simulate each system at neutral and acidic pH in triplicate for a total of 30 systems. The pH of the system is controlled by the protonation state of the Asp residues; Acidic pH (2-4) is achieved by protonating the functional group of the Asp residues while neutral pH leaves the Asp residues deprotonated giving each β-strand a total charge of 0 and +4, respectively.</p><p>The C-termini remain deprotonated at both acidic and neutral pH.</p><p>We simulate five coumarin-peptide systems to ascertain the stability and optoelectronic properties of coumarin fibers with varying hydrophobicity. An unnatural coumarin amino acid is substituted for the fourth hydrophobic residue in the (RXDX) 4 sequence ((RXDX)-RXD-Coumarin-(RXDX) 2 ). Analogous to the (RXDX) 4 fiber simulations, each coumarin-RXDX fiber is simulated at both neutral and acidic pH in triplicate. Coumarin and an unnatural amino acid coumarin are parametrized in ff15ipq using the IPolQ method 64,65 with the ωB97X-D3 functional 66,67 and the cc-pVTZ basis set. 68 Atom types and charges are provided in the SI. QM calculations were run using ORCA 3.1.3. 69 All molecular dynamics simulations are run using the AMBER18 software 62 and modeled using AMBER's ff15ipq 64,65 and GAFF force fields. 70 The choice in force field is verified by comparison of fiber thickness with experimental values provide in Table 1.</p><!><p>Both the (RADA) 4 and (RLDL) 4 systems are found to have thicknesses within error of experiment. Furthermore, the calculated solubility of our coumarin model is 0.014 ± 0.006 mol L , in agreement with the solubility value of 0.01706 mol L reported by Yalkowsky et al. 71 Hydrogen atoms are constrained using the SHAKE algorithm. 72 Direct nonbonding interactions are cutoff at 12 Å and long range electrostatic interactions are modeled using the particle mesh Ewald treatment (PME). 73 An integration time step of 2 fs is used. MD simulations are performed in the NPT-ensemble with a Monte Carlo barostat set to 1 atm and a Langevin thermostat set to 298 K.  28 -23(4) 28 --</p><p>The simulation protocol used for all systems is the same. Solvated systems are minimized in two steps: 8,000 steepest descent minimization steps with harmonic constraints (force constant of 100 kcal mol −1 Å−2 ) placed on all peptide atoms, followed by 12,000 conjugate gradient steps with no constraints. The system is heated to a temperature of 298K over 50 ps with harmonic restraints (force constant of 5 kcal mol −1 Å−2 ) on all peptide atoms. The water in the system relaxes with a harmonic restraint (force constant of 5 kcal mol −1 Å−2 ) on all solute atoms for 40 ps. Following the heating protocol, the system is simulated at 298K for 10 ns allowing the pressure to equilibrate. For the (RXDX) 4 and coumarin-(RXDX) 4 simulations we use simulated annealing molecular dynamics (SAMD) due to the slow time dynamics of the fiber systems. Thirty simulated annealing cycles with a period of 10.05 ns were performed.</p><p>In each cycle, the temperature is increased from 298 to 450 K in 50 ps, then kept constant for 1 ns, subsequently lowered back to 298K in 8.0 ns, and finally kept constant for 1 ns. The system is simulated at 298K for 200 ns from which ensemble averages were measured. The combined simulation time for this study is 31.25 µs.</p><!><p>Previous studies of the RXDX peptide sequence (X=A,L) have shown that the RXDX peptides form β-sheet sandwich fibers at neutral pH that dissociate in acidic and basic environments. [24][25][26][27]29,63 Experimental studies suggest that hydrophobicity is the main driving force for fibril formation rates and stability. It was proposed that aromaticity is not necessary for fibril formation, but does affect self-assembly in these materials as changes in peptide and ion concentrations lead to different fiber morphologies that are not seen when other non-aromatic non-polar residue were used. Here, we present a set of simulations that investigate the effect hydrophobicity and aromaticity have on the stability and switchability of (RXDX) 4 fibers.</p><p>We present a similar set of simulations with the addition of an unnatural coumarin amino acid to the (RXDX) 4 sequence as a model system for the design of switchable self-assembling biomaterials with interesting optoelectronic properties. These simulations probe the stability and pH-switchability of the the β-sheet sandwich structure, as well as characterize the crucial role hydrophobicity plays in the stability of the fiber.</p><p>Furthermore, we study the pH-switchability of the coumarin order which determines the optical and electronic properties of coumarin-(RXDX) 4 fibers.</p><!><p>Changing the pH of the (RXDX) 4 systems from neutral to acidic leads to instability within the β-sheet fibers. Representative snapshots of the (RADA) 4 simulations at neutral and acidic pH are shown in the first column of Figure 3(a). At neutral pH, the system retains the β-sheet sandwich structure for the entire simulation other than some end fraying. Under acidic pH, the system completely loses all β-sheet structure leading to all of the peptides being dispersed in the solvent. This behavior is quantified for the entire simulation by measuring the percent of residues in an anti-parallel β-sheet, Secondary structure was calculated using the DSSP method as a measure of fiber stability. Fibers become more stable with increasing hydrophobicity at both pH leading to a reduction in the pH-switchability of the fiber structure.</p><p>Mutation of the RADA peptide sequence at the alanine position with increasingly hydrophobic residues enhances fiber stability and diminishes the pH-switchability.</p><p>Representative snapshots of the (RXDX) 4 (X = V, L, I, F) simulations are provided in Figure 3(a). Generally, the fibers at neutral pH seem relatively unaffected by the identity of the hydrophobic residue in the RXDX sequence. This is supported by the 50-70% of residues residing in a β-sheet for the course of these simulations as quantified in Aromaticity of the nonpolar residues plays a minor roll in β-sheet fiber stability.</p><p>It has been argued that aromaticity is unnecessary for fibrillization, instead, it is primarily hydrophobicity that influences fibrilization; a peptide sequence needs to reach a hydrophobic threshold to fibrilize. 56 Figure 3 shows that fibers containing Phe are more stable than fibers containing Ile, even though Phe and Ile have the same hydrophobicity and Ile has a higher propensity to form β-sheets (Table S1).</p><p>To demonstrate that aromaticity further stabilizes (RXDX) 4 fibers due to π-stacking interactions between aromatic sidechains we calculate the free energy as a function of the angle between neighboring phenyl rings separated into inter-sheet and intra-sheet pairs for the (RFDF) 4 systems. As shown in Figure 4, there is a preference for the phenyl rings to π-stack within a β-sheet as there is an approximately 3 kcal/mol well at an angle of 0°for intra-sheet pairs. Interestingly, there is no intercalation of the phenyl rings between β-sheets. The phenyl rings sit end-to-end in a T-shape configuration with an angle of 58°as shown by the inter-sheet free energy curve. These data support the conclusion that hydrophobicity is an important factor in determining fiber stability, but additionally suggest that the planar geometry of the phenyl ring further stabilizes (RXDX) 4 fibers at both neutral and acidic pH. The increase in stability for the (RFDF) 4 fiber could originate from aromatic interactions between phenyl rings, the packing of the phenyl rings within the fiber, or a more favorable change in entropy during self-assembly due to the more restricted range of motion of the Phe sidechain structure relative to the Ile sidechain structure before self-assembly. Free energy of the dihedral between phenyl rings in the (RFDF) 4 fiber separated into intra-and inter-sheet pairs. The free energy minima at 0°for intra-sheet pair demonstrate π-stacking interactions within each β-sheet while inter-sheet phenyl ring pairs form a T-shape configuration with a dihedral of 58°.</p><!><p>Coulombic interactions between residues within the fiber are responsible for the pHswitchability of the (RXDX) 4 fibers. The only difference between the neutral and acidic pH systems is the protonation state of the Asp residue leading to a change in the coulombic interactions stabilizing the fiber. Figure 5a contains the coulombic interactions of (RADA) 4 averaged over the first 10 ns of the simulation broken down in to residue type pairs for the initial self-assembled structure. There are a mix of stabilizing and destabilizing electrostatic interactions. The Arg-Arg, Asp-Asp, and X-X interactions are large and destabilize the fiber's β-sheet sandwich structure. These interactions are counterbalanced by the Arg-Asp, X-Asp, and X-Arg stabilizing interactions. At neutral pH, the total coulombic interactions are large and negative, stabilizing the fiber.</p><p>When the pH is lowered both the Asp-Asp and Arg-Asp coulombic interactions are significantly reduced due to the protonation of the aspartates to aspartic acids. Since the Arg-Asp interactions are 3-4 times larger than the Asp-Asp interactions this leads to an overall 9,870 kcal/mol increase in the total coulombic interaction energy. It is the large reduction of the Arg-Asp stabilizing interaction energy which leads to a destabilization of the (RADA) 4 fiber at acidic pH. Furthermore, the large change in coulombic interaction energy is consistent for all 5 (RXDX) 4 fibers and is independent of the identity of the hydrophobic residue.</p><p>Increasing the hydrophobicity of (RXDX) 4 peptides leads to an enhanced fiber stability and a reduction in pH-switchability due to an increase in hydrophobic interactions within the fiber. Hydrophobic molecules aggregate in water because this maximizes the number of hydrogen bonds between water molecules and minimizes the contact area between the hydrophobic and water molecules. Therefore, the free energy of solvation of hydrophobic molecules is negatively correlated to the molecule's solvent accessible surface area (SASA). The change in hydrophobic SASA of (RXDX) 4 molecules during self-assembly is used as a measure of the hydrophobic interactions within each fiber.</p><p>The hydrophobic SASA is calculated as the total SASA of the hydrophobic residues in the (RXDX) 4 peptides. Figure 5b shows the total change in SASA of the hydrophobic residues due to the self-assembly of (RXDX) 4 peptides into β-sheet fibers. As expected, there is a larger change in hydrophobic SASA as the hydrophobicity of the peptide is increased at both pHs. This suggests there is a subtle balance between the coulombic and hydrophobic interactions controlling the stability of (RXDX) 4 fibers. For (RADA) 4 the hydrophobic interactions are small enough that the reduction in stabilizing coloumbic interactions with pH change lead to instability within the fiber. As the non-polar residue is mutated to a more hydrophobic residue, the balance between coulombic and hydrophobic interactions shift such that the hydrophobic interactions are large enough to stabilize the fiber even at acidic pH. The mutation of a single hydrophobic residue within the (RXDX) 4 sequence to coumarin leads to changes in fiber stability, pH-switchability, and structure. Addition of coumarin leads to an induced curvature and a destabilization of the β-sheet fiber due to differences in the size of the hydrophobic residue and the coumarin side chain. Figure 6b shows the percent of residues in a β-sheet. (RADA) 4 fibers are completely unstable with the addition of coumarin making it a poor candidate for the design of switchable electronic biomaterials. The other systems are able to retain 40-70% of the β-sheet structure. Figure 6a shows each of the coumarin-(RXDX) 4 fiber structures. Even though the systems retain most of their β-sheet content with pH change, the overall fiber structure is unstable either breaking into smaller β-sheets containing only a few peptide strands or the two β-sheets that make up the fiber separate exposing the hydrophobic layers to the solvent. Although the pH-switchability of the fibers are not shown in the β-sheet metric, there is a change in stability and structure of the fiber with pH. The fibers containing coumarin are more stable with increasing hydrophobicity as the hydrophobic residues become similar in size to the coumarin sidechain.</p><p>Increasing the hydrophobicity and size of the nonpolar residue decreases the curvature of the fiber structure and increases fiber stability. Figure 6d and Figure 6e show the distribution of the bend angle for each system at neutral and acidic pH, respectively.</p><p>The bend angle is a measure of the curvature of the fiber in the direction of fibrilization and is calculated using the method described by Fujiwara et al. 75 At neutral pH the (RFDF) 4 fiber has very little curvature as the bend angles are close to zero. As the hydrophobicity of the peptide increases, the bend angle distribution broadens out and shifts to the right corresponding to less order in the fiber and an increase in fiber curvature. Furthermore, lowering the pH leads to increased fiber curvature and more disorder in the fiber as the distributions broaden and shift to higher angles.</p><p>The total SASA of the coumarin residues within each fiber provide further evidence of an enhanced fiber stability with increasing hydrophobicity of the coumarin-(RXDX) 4 sequence. For an ordered fiber, the coumarin SASA is small as only the coumarins at the end of the fiber will be solvent exposed, but as the fiber dissociates, more coumarins become solvent exposed. Figure 6c shows the average coumarin SASA for each fiber at both neutral and acidic pH. In agreement with the trends observed in the bend angle analysis, there is a decrease in coumarin SASA and therefore, an increase in fiber stability as the hydrophobicity of the peptide increases. When the pH is lowered the coumarin SASA increases corresponding to a decrease in fiber stability.</p><!><p>As part of designing a switchable material, it is important to not only have pHswitchability of the coumarin-(RXDX) 4 fiber structure, but also in the the optical properties of the fiber. For this reason it is of interest to analyze the coumarin order within the fibers because it is the arrangement of the coumarin sidechains that will dictate the optical and electronic properties of the fiber. The addition of coumarin to the (RADA) 4 sequence leads to instability and dissociation of the fiber making coumarin-(RADA) 4 a poor candidate for the design of switchable electronic biomaterials. Therefore, we analyze the pH-switchability of the coumarin order within (RVDV) 4 , (RLDL) 4 , (RIDI) 4 , and (RFDF) 4 fibers to discern which of these sequences are the most optimal for these materials. The joint probability density as a function of both the coumarin-coumarin separation distance and the angle between the dipole moments of the two coumarin is used to measure the organization of coumarin within coumarin-(RXDX) 4 fibers. Furthermore, we use relative entropy to quantify the pHswitchability of the structure of the coumarin network. Relative entropy is a measure of the difference between two probability densities. The relative entropy between the joint probability densities of the neutral and acidic systems for each fiber is calculated as</p><p>where P n (r, θ) and P a (r, θ) are the joint probability densities of the neutral and acidic system, respectively. Coumarin sidechains become more ordered within the β-sheet fibers with increasing peptide hydrophobicity. Figure 7 shows the joint probability distribution for (RLDL) 4 , (RIDI) 4 , and (RFDF) 4 systems at neutral and acidic pH. At neutral pH there is a high degree of coumarin order for the (RFDF) 4 fibers as there are large peaks near 0°that repeat out to long distances corresponding to a bricklayer structure composed of a majority of the coumarins within the fiber. (RLDL) 4 shows little coumarin order as there is a broad distribution of angles between coumarin sidechain and little density at large distances. Generally, as the hydrophobicity, size, and aromaticity of the peptide increases there is a shift to a parallel arrangement of the coumarins and an increase in density at larger distances corresponding to larger coumarin aggregates within the fiber. When the pH is lowered there is a reduction in long range structure and the distribution of angles between coumarin dimers broadens. This is most apparent in the coumarin-(RFDF) 4 fibers where there is a significant increase in sampling of angles near 90°and a reduction in sampling of angles near 0°. Modifying the hydrophobicity of the nonpolar residue alters the pH-switchability of coumarin order within coumarin-(RXDX) 4 fibers. Table 2 shows the relative entropy between the neutral and acidic joint probability densities for each fiber system.</p><p>Contrary to the trends in pH-switchability of (RXDX) 4 and coumarin-(RXDX) 4 fiber structures, there is an increase in pH-switchability of coumarin ordering with increasing hydrophobicity of the peptide sequence, where coumarin-(RFDF) 4 fibers have the largest relative entropy between the neutral and acidic probability densities. These results suggest that for the development of switchable self-assembling biomaterials using Coumarin-(RXDX) 4 sequences, coumarin-(RFDF) 4 is the most ideal as it shows the largest pH-switchability of the coumarin sidechain order and therefore, the largest change in the optical and electronic properties of the fiber.</p><!><p>We simulate a series of (RXDX) 4 and coumarin-(RXDX) 4 β-sheet sandwich fibers to investigate the role of hydrophobicity in the stability and pH-switchability of the fibers for the development of switchable self-assembling biomaterials with interesting optical and electronic properties. Generally, the fibers are shown to be pH switchable as they became destabilized at acidic pH. Increasing the hydrophobicity of the peptide sequence leads to an increase in fiber stability at both neutral and acidic pH resulting in a decrease in pH-switchability. Addition of coumarin to the (RXDX) 4 sequence results in fiber instability and induces a curvature in the fiber structure due to differences in the size between the coumarin sidechains and the hydrophobic amino acid sidechains.</p><p>These effects are reduced at neutral pH relative to acidic conditions and with larger and more hydrophobic sidechains. Coumarin sidechains become more ordered and showed the largest pH-switchability with increasing hydrophobicity. Of the systems we studied the coumarin-(RFDF) 4 sequence was the most stable fiber with the addition of coumarin into the sequence and the fiber was most stable and had the largest change in the organization of coumarin with pH change making it the most ideal for these types of materials. Future work will measure the optical and electron transfer properties of these fibers as well as investigate the self-assembly mechanism of these materials.</p><!><p>Force field verification data, coulombic interaction energies decomposed into residue type pairs, and coumarin ordering joint probability densities not presented here.</p>