Assessing the Utility of Infrared Spectroscopy as a Structural

Article pubs.acs.org/Langmuir

Assessing the Utility of Infrared Spectroscopy as a Structural Diagnostic Tool for β‑Sheets in Self-Assembling Aromatic Peptide Amphiphiles Scott Fleming,†,§ Pim W. J. M. Frederix,†,‡,§ Iván Ramos Sasselli,†,§ Neil T. Hunt,‡ Rein V. Ulijn,*,† and Tell Tuttle*,† †

WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, U.K. Department of Physics, University of Strathclyde, SUPA, Glasgow G4 0NG, U.K.

‡

S Supporting Information *

ABSTRACT: β-Sheets are a commonly found structural motif in self-assembling aromatic peptide amphiphiles, and their characteristic “amide I” infrared (IR) absorption bands are routinely used to support the formation of supramolecular structure. In this paper, we assess the utility of IR spectroscopy as a structural diagnostic tool for this class of self-assembling systems. Using 9-fluorene-methyloxycarbonyl dialanine (Fmoc-AA) and the analogous 9-fluorene-methylcarbonyl dialanine (Fmc-AA) as examples, we show that the origin of the band around 1680−1695 cm−1 in Fourier transform infrared (FTIR) spectra, which was previously assigned to an antiparallel β-sheet conformation, is in fact absorption of the stacked carbamate group in Fmoc-peptides. IR spectra from 13 C-labeled samples support our conclusions. In addition, DFT frequency calculations on small stacks of aromatic peptides help to rationalize these results in terms of the individual vibrational modes.

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INTRODUCTION Infrared (IR) spectroscopy is well-established as a useful technique to assist in the determination of secondary structure elements in proteins, specifically in the amide I region (1600− 1700 cm−1), which is very sensitive to hydrogen-bonding patterns found in α-helices and β-sheets.1−7 Recently, IR spectroscopy has been utilized to help characterize the supramolecular structure of self-assembling hydrogels composed of oligopeptides. It is theoretically possible to differentiate between infinite parallel and antiparallel β-sheets (vide inf ra)the former typically showing a single band at approximately 1615−1640 cm−1 and the latter having an additional component near 1685 cm−1. This practice of using models meant for the secondary structure determination of proteins may not be valid for interpreting the infrared spectra of these aromatic di- and tripeptide hydrogels. In examples that consist of 7−29 amino acid residues,8−11 typically a 1615 cm−1 amide I peak and a much weaker 1680 cm−1 peak are observedcharacteristic of proteins with an antiparallel β-sheet structure. However, the presence of β-sheets has also been reported for a class of low molecular weight (LMW) gelators composed from various short (e.g., di- and tri-) peptides capped at the N-terminus with an aromatic groupmost commonly the Fmoc moiety (Fmoc, 9-fluorenylmethyloxycarbonyl).12−21 The assignment of infrared bands that are apparently analogous to those seen in longer © XXXX American Chemical Society

peptides is often used as a key piece of evidence to support an antiparallel β-sheet structure. While the absorption bands of LMW gelators resemble β-sheet signals in terms of line position, the relative infrared peak intensities for these materials are not always typical of that found for longer peptides or indeed proteins.22 Significant variation in the intensity of the 1690 cm−1 band has been observed, with this band occasionally being of similar or greater magnitude compared to the lower frequency (1615−1640 cm−1) amide I contribution,14,19,20,23,24 which is higher than expected for even a perfect antiparallel βsheet.4−6 Additionally, the relative intensity of the 1690 cm−1 band is observed to decrease as the ratio of amide to carbamate groups increases in longer Fmoc-peptide examples.18,21 This is not consistent with what would be expected if this peak is truly indicative of an antiparallel β-sheet in these systems, especially since the longer chain length would be more likely to increase the ν∥ component (see below), rather than decrease it. The ambiguity surrounding these assignments has led to doubt regarding the presence of antiparallel β-sheet structures in spite of infrared evidence suggesting this conformation, with authors increasingly wary of applying traditional protein secondary structure interpretations.25−27 In spite of IR evidence for Received: March 20, 2013 Revised: June 11, 2013

A

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hydrogels (Fmc, fluorenylmethylcarbonyl), both experimentally using FTIR spectroscopy and isotope labeling, and theoretically employing density functional theory (DFT). Dialanine was chosen as the model dipeptide as it is the smallest chiral amino acid, which helped to facilitate the computationally demanding simulations. However, the calculation of amide I modes of a certain peptide is not significantly influenced by the nature of the side chains, so the approach presented here is expected to be valid for all short peptides (although the optimized supramolecular structure and hence the resulting spectrum may differ with different peptides). Fmc was utilized as a close analogue for the Fmoc moiety but crucially lacks the carbamate oxygen and is instead linked to dialanine via an amide bond (see Figure 1). Additionally, we will briefly discuss the suitability of DFT frequency calculations for predicting IR spectra of self-assembling systems.

“antiparallel” β-sheets, parallel β-sheet structures have been proposed based on X-ray diffraction data,26 and a recent experimental and computational study has suggested a polyproline II conformation,27 which lacks significant hydrogen bonding between residues. Additionally, we have previously observed the absence of the 1690 cm−1 peak,28 in hydrogels assembled from non-Fmoc aromatic dipeptides that lack the carbamate group. The carbamate group is known to absorb IR light in the 1685− 1730 cm−1 range,29−33 including a report by Nuansing et al. on Fmoc-FG powders,34 significantly higher than the 1650 cm−1 absorption for free amide groups. For these reasons, it is important to assess the diagnostic value of the amide I infrared region for these systems. In the general case of antiparallel β-sheets, the low-frequency component (1615−1640 cm−1) arises from interstrand delocalization and mode coupling (ν⊥, see Figure 1), which

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EXPERIMENTAL SECTION

All reagents were purchased from commercial sources and were used as supplied, unless stated otherwise in the experimental procedures. Details of synthesis and analysis are available in the Supporting Information. Formation of Gels. 20 mM deuterated gels were formed by dispersing Fmoc or Fmc in deuterium oxide (1 mL) and dissolving via the addition of 0.5 M NaOH deuterium oxide solution (50 μL). 1 M HCl was then added dropwise while the samples underwent repeated sonication and vortexing. The samples were self-supporting upon vial inversion at pH ∼ 4−5 (pH measured in an H2O sample). Only broad, baseline-type absorptions from HOD or H2O were observed in the amide I region. The same preparation method was used for isotope labeled 13C-labeled Fmoc* gel. Methanol solutions of Fmoc or Fmc were prepared at the same peptide concentration. Fourier Transform Infrared Spectroscopy. Spectra were recorded on a Bruker Vertex 70 spectrometer averaging 25 scans per sample at a resolution of 1 cm−1. Samples were transferred into a standard IR cell holder (Harrick Scientific) and sandwiched between two 2 mm CaF2 windows separated with a 25 μm PTFE spacer. No significant loss of water was observed during sample preparation. Vibrational Frequency Calculations. Molecules were modeled in their C-terminally protonated form, since spectroscopy has previously shown that within hydrogels the carboxylic acid groups exist predominantly in this forma consequence of the apparent pKa shift of the carboxylic acids when in the self-assembled state.22,23 All exchangeable hydrogens in the system were 1H, which has been shown to have a negligible effect on amide I frequencies.36 Gas phase structure optimizations were performed using Grimme’s B97-D functional38 to take dispersion effects into account, which are important for geometry optimizations in systems with an aromatic group like fluorenyl, with the def2-SVP basis set39 taking advantage of the MARI-J approximation40 within the Turbomole 6.3.1 program.41 As this functional is not available in Turbomole for frequency calculations, these were performed in Gaussian09.42 No scaling factor was applied to the results to correct for anharmonic effects and basis set truncation. Vibrations were visualized using the TmoleX program.43 Vibrational modes located >50% (by magnitude of normal mode vectors on relevant carbon atoms) on moieties other than the amide(s) or carbamate were omitted from the simulated spectra. Labels 1 and 2 indicate the number of the carbonyl giving the main contribution to the transition dipole moment (see labeling in Figure 1a,b) determined by the largest local displacement of the carbonyl group. Modes with significant (20−50%) contribution from carboxylic acid groups are indicated with a dagger (†), while amide I modes arising from amide groups with their carbonyl group pointing out of the stack are indicated with an asterisk (∗). For both of these, the calculated amide vibrational frequency is 30−100 cm−1 higher than would be expected due to combination with a higher frequency mode

Figure 1. Fmoc-AA and Fmc-AA. (a, b) Chemical structures, with numbered carbonyl groups. The asterisk in (a) indicates the position of the 13C in the corresponding isotope labeled structure. (c−f) Dimeric molecular models showing the repeated β-sheet hydrogen bonding patterns for (c) parallel Fmoc, (d) parallel Fmc, (e) antiparallel Fmoc, and (f) antiparallel Fmc. Arrows show the direction of parallel (||) and perpendicular (⊥) excitons as discussed in the text.

shifts the mode to lower wavenumbers as strands become more aligned and the number of strands per sheet increases.4,5 In contrast, the frequency of the high wavenumber peak (1680− 1695 cm−1) is generally independent of the number of strands and originates from vibrational excitons that run along a particular β-strand (ν∥, Figure 1).4 As a consequence, the transition dipole moment along the strands will be relatively small for short peptide β-sheet strands (≤3 amino acids), and it is conceivable that this peak may not be resolvable in spectra of their supramolecular structures. Moreover, in cases of β-sheets of finite size, correctly determining the presence of a parallel or antiparallel structure can be challenging; as disorder or twists, which may be expected for short, flexible peptides, can severely diminish the delocalization of vibrational modes and therefore broaden or shift optical transitions.1,35−37 In this paper we study the amide I infrared bands of Fmocdialanine27 (from here on: Fmoc) and Fmc-dialanine (Fmc) B

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or lack of the cooperative dipole coupling and hydrogen bonding, respectively.1

the latter is expected to be of negligible intensity for very short peptide chains. This is confirmed by the spectra of Fmoc and Fmc in neat methanol at the same concentration (Figure 2a). Despite the absence of self-assembly in this solvent, it is clear that the spectrum of Fmoc shows distinctly separate bands at 1669 and 1708 cm−1 assigned to the respective amide I and carbamate absorptions, whereas Fmc shows two overlapping amide I bands around 1652 and 1670 cm−1 from carbonyl 1 and 2, respectively. Furthermore, in both experimental spectra a band is present at 1730 cm−1 due to the carboxylic acid terminal group. Note that a weak MeOH absorption is responsible for the sloped background below 1600 cm−1. It is apparent from Figure 2a that the absorption of Fmoc and Fmc in methanol solution has a much lower intensity due to the lack of the cooperative effect (note the difference in scale between Figures 2a and 2b), but the relative intensities of the bands are preserved. This indicates that, in the hydrogel, both the 1638 cm−1 amide band and the 1685 cm−1 carbamate band of Fmoc are equally enhanced as part of the β-sheet structure, which again suggests that interstrand delocalization is responsible in each case, rather than ν∥ and ν⊥ components. Conclusive evidence for the assignment of the 1685 cm−1 peak to the carbamate group was given by the IR spectrum of isotope-labeled Fmoc* hydrogels, in which a single 13C substitution was made at the amide carbonyl (see Figure 1a). This procedure typically red-shifts the vibrational frequency of the H-bonded carbonyl groups by 40−43 cm−1 (e.g., ref 46). Figure 2b shows that, in comparison with unlabeled Fmoc, only the lower frequency band moves from about 1638 to 1597 cm−1, while the position of the 1685 cm−1 peak remains unaffected, in agreement with its different chemical nature. DFT Calculations. Vibrational frequencies were calculated for the discrete molecules (see Figure 3a). Despite the absence of any β-sheet structure, the monomer of Fmoc shows two distinct peaks with a separation of about 52 cm−1, while Fmc demonstrates a much smaller separation of 17 cm−1. Moreover, when looking at the origin of the bands and assigning them to the carbonyl group with the largest contribution to the vibration (for labeling see Figure 1a,b), it becomes apparent that the carbamate carbonyl (label 1 in Fmoc spectra) vibrates at a distinctly higher frequency (67 cm−1) than the analogous Fmc-amide group (1 in Fmc spectra). The amide groups present in the middle of both monomers (labeled 2) are predicted to have similar frequencies, as expected. Next, parallel and antiparallel dimers and tetramers of both Fmoc and Fmc were used to mimic the potential supramolecular structures (Figure 1c−f). In addition, two alternative antiparallel arrangements were considered (see Supporting Information, Figures S1 and S2). For the antiparallel and parallel tetramers (Figure 3b,c) and other intermolecular conformations (see Figures S4−S9), both Fmoc and Fmc spectra show multiple normal modes. This introduction of new peaks is expected as the number of oscillators in the system increases; however, experimentally many of the bands would overlap due to the line broadening associated with aqueous solvation. One would expect that the parallel (ν∥) and perpendicular (ν⊥) modes should progressively increase in intensity due to the coupling and cooperativity associated with these modes in an extended antiparallel β-sheet form. Nonetheless, the additional modes will also be present experimentally (i.e., as a weak, broad component around 1650 cm−1), even in near-perfect sheets.5

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RESULTS AND DISCUSSION FTIR Spectroscopy and Isotope Labeling. Hydrogels were prepared via the dropwise addition of HCl to 20 mM solutions of Fmoc and Fmc in D2O at an initial pH of ∼9 and a final pH of ∼4−5. The FTIR spectrum of the Fmoc gel (Figure 2b) clearly shows the characteristic bands described previously,

Figure 2. Experimental infrared spectra of Fmoc and Fmc as (a) methanol solutions and (b) deuterated hydrogels.

centered around 1638 and 1685 cm−1. Both bands are of a similar intensity, which is not as expected for antiparallel βsheets, but is characteristic for Fmoc-dipeptide hydrogels.22 In contrast, the gel of Fmc shows only a single main peak at 1626 cm−1, although the non-Gaussian shape suggests this is likely the convolution of two close absorption bands (i.e., amide groups no. 1 and 2 in Figure 1b). A small peak around 1650 cm−1 is usually attributed to “random coil” conformation in protein spectroscopy. In short peptides, this is more commonly assigned to disordered44 or unstacked45 amide groups, indicating imperfect sheet stacking or partial polyproline II conformation.27 However, the dominant high-frequency peaks at 1638 cm−1 (Fmoc) and 1626 cm−1 (Fmc) are more consistent with β-sheet structure.22 In both spectra, a broad contribution around 1590 cm−1 is discernible, which indicates that a fraction of terminal carboxylic acid groups remain deprotonated, in line with other Fmoc-dipeptide gels.22,45 Assuming that the self-assembly of Fmoc and Fmc proceeds in a similar fashion, these results would indicate the 1685 cm−1 peak is more likely a consequence of carbamate moiety absorption than the ν∥ component of antiparallel β-sheets, as C

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average modes arising from the amide group labeled 1 absorb at a lower frequency (1679 vs 1688 cm−1). It should be noted that some modes, like the 1783 cm−1 band in Figure 3b (indicated with a dagger), contain a significant contribution (28%) from a carboxylic acid vibration, which can shift the vibrations toward higher frequencies by ∼30 cm−1. Furthermore, in all calculated spectra, the outermost C O groups at the periphery of a β-sheet are predicted to absorb IR at higher frequencies due to the lack of H-bonding, as marked by asterisks in Figure 3b,c and Figures S4−S9. These peaks can be ignored for our purposes as the contribution from the edge of the stack will diminish in larger systems but could show up in experimental spectra of disordered secondary structures as part of a component around 1650 cm−1, typically assigned to random coil peptides. In summary, although there are clearly size-dependent artifacts in the model systems, these can be easily identified and rationalized. The DFT results for both monomers and supramolecular stacks support the notion that the presence of the “split” amide I peak in systems based on Fmoc is more dependent upon the presence of a two different chemical moieties (i.e., carbamate and amide) rather than indicative of an antiparallel β-sheet structure. As well as vibrational calculations, binding energies were also computed for these structures (see Figure S3). β-Sheets of Fmc were found to be more stable than Fmoc, and the tetramers were more stable (on a per monomer basis) than the corresponding dimers due to cooperative binding effects. In addition, the parallel arrangement was generally found to be more stable than the antiparallel arrangement. However, the small variations in relative binding energies of these arrangements are not good indicators for determining the equilibrium distribution of supramolecular structures due to the neglect of several important features within these simplified models (e.g., explicit solvent, edge effects, etc.). Rather, the correlation between the experimental and calculated vibrational spectra is a much more reliable indicator due to the local nature of the factors effecting the shifts in the amide I bands. When the calculated infrared bands (Figure 3) are compared with the experimental results (Figure 2), several key features become apparent. When ignoring size-dependent artifacts in our DFT results, the line shapes of the predicted absorption bands are in good agreement with experimental results. Furthermore, the experimental results consistently show the amide I peak of Fmc at a lower frequency than that seen for the infrared bands of Fmoc. This observation suggests the proximity of the fluorenyl group also influences the amide I vibration in agreement with the computational spectra: amide group 1 in Fmc absorbs at a lower frequency than amide groups 2 in both Fmc and Fmoc. Although the calculated frequencies are systematically too high, it is apparent that, as the finite computational models become larger, a gradual convergence toward the experimental infrared bands is observed (see Figure 3 and Figures S4−S9). It should be noted that our frequency results have not been corrected for anharmonic effects and basis set truncation, which usually leads to an overestimation of the frequencies, because no extensively tested scaling factor is known for the combination of B97-D functional and def2-SVP basis set. By applying a standard scaling factor (e.g., 0.97), the agreement between the calculated and experimental data would be further improved. Furthermore, the antiparallel tetramer results (Figure 3b) appear to more closely resemble the experimental infrared spectra (Figure 2) when compared with the analogous parallel

Figure 3. Simulated amide I spectra for Fmoc and Fmc (a) monomers and (b) antiparallel and (c) parallel tetramers. Spectra are generated by applying an 8 cm−1 Gaussian line width49 to the calculated normal modes (vertical drop lines). Labels 1 and 2 indicate the number of the carbonyl giving the main contribution to the transition dipole moment (see labeling in Figure 1a,b). Vibrations localized on carboxylic acid groups (carbonyl 3, generally >1790 cm−1) groups are omitted. The mode indicated with a dagger (†) has significant contribution from a carboxylic acid group, while amide I modes arising from amide groups with their carbonyl group pointing out of the stack are indicated with an asterisk (∗).

In our calculated spectra we observe a cooperative increase in IR absorption coefficients with growing stack sizes. Additionally, we see two main groups of Fmoc bands: carbamate and amide groups are predicted to absorb at different frequencies (see Figure 3 and Figures S4−S9). When the line positions for all the tetramer configurations are weighted by the intensity of their modes, the average predicted absorption frequencies are 1742 and 1699 cm−1 for Fmoc modes mainly located on carbamate and amide groups, respectively, showing a significant 43 cm−1 separation. In contrast, in the calculated Fmc spectra peaks from both amide groups are closer together, although on D

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(2) Rughani, R. V.; Schneider, J. P. Molecular design of beta-hairpin peptides for material construction. MRS Bull. 2008, 33, 530−535. (3) Yan, H.; Frielinghaus, H.; Nykanen, A.; Ruokolainen, J.; Saiani, A.; Miller, A. F. Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway. Soft Matter 2008, 4, 1313−1325. (4) Lee, C.; Cho, M. Local amide I mode frequencies and coupling constants in multiple-stranded antiparallel β-sheet polypeptides. J. Phys. Chem. B 2004, 108, 20397−20407. (5) Hahn, S.; Kim, S.-S.; Lee, C.; Cho, M. Characteristic twodimensional IR spectroscopic features of antiparallel and parallel βsheet polypeptides: Simulation studies. J. Chem. Phys. 2005, 123, 084905−084905−10. (6) Ganim, Z.; Chung, H. S.; Smith, A. W.; DeFlores, L. P.; Jones, K. C.; Tokmakoff, A. Amide I two-dimensional infrared spectroscopy of proteins. Acc. Chem. Res. 2008, 41, 432−441. (7) Cheatum, C. M.; Tokmakoff, A.; Knoester, J. Signatures of βsheet secondary structures in linear and two-dimensional infrared spectroscopy. J. Chem. Phys. 2004, 120, 8201−8215. (8) Rughani, R. V.; Salick, D. A.; Lamm, M. S.; Yucel, T.; Pochan, D. J.; Schneider, J. P. Folding, self-assembly, and bulk material properties of a de novo designed three-stranded β-sheet hydrogel. Biomacromolecules 2009, 10, 1295−1304. (9) Measey, T. J.; Schweitzer-Stenner, R. Aggregation of the amphipathic peptides (AAKA)n into antiparallel β-sheets. J. Am. Chem. Soc. 2006, 128, 13324−13325. (10) Measey, T. J.; Smith, K. B.; Decatur, S. M.; Zhao, L.; Yang, G.; Schweitzer-Stenner, R. Self-aggregation of a polyalanine octamer promoted by its C-terminal tyrosine and probed by a strongly enhanced vibrational circular dichroism signal. J. Am. Chem. Soc. 2009, 131, 18218−18219. (11) Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajagopal, K.; Pakstis, L.; Kretsinger, J. Responsive hydrogels from the intramolecular folding and self-assembly of a designed peptide. J. Am. Chem. Soc. 2002, 124, 15030−15037. (12) Shao, H.; Parquette, J. R. A pi-conjugated hydrogel based on an Fmoc-dipeptide naphthalene diimide semiconductor. Chem. Commun. 2010, 46, 4285−4287. (13) Orbach, R.; Mironi-Harpaz, I.; Adler-Abramovich, L.; Mossou, E.; Mitchell, E. P.; Forsyth, V. T.; Gazit, E.; Seliktar, D. The rheological and structural properties of Fmoc-peptide-based hydrogels: the effect of aromatic molecular architecture on self-assembly and physical characteristics. Langmuir 2012, 28, 2015−2022. (14) Hirst, A. R.; Roy, S.; Arora, M.; Das, A. K.; Hodson, N.; Murray, P.; Marshall, S.; Javid, N.; Sefcik, J.; Boekhoven, J.; Van Esch, J. H.; Santabarbara, S.; Hunt, N. T.; Ulijn, R. V. Biocatalytic induction of supramolecular order. Nat. Chem. 2010, 2, 1089−1094. (15) Xu, H. X.; Das, A. K.; Horie, M.; Shaik, M. S.; Smith, A. M.; Luo, Y.; Lu, X. F.; Collins, R.; Liem, S. Y.; Song, A. M.; Popelier, P. L. A.; Turner, M. L.; Xiao, P.; Kinloch, I. A.; Ulijn, R. V. An investigation of the conductivity of peptide nanotube networks prepared by enzyme-triggered self-assembly. Nanoscale 2010, 2, 960−966. (16) Jayawarna, V.; Richardson, S. M.; Hirst, A. R.; Hodson, N. W.; Saiani, A.; Gough, J. E.; Ulijn, R. V. Introducing chemical functionality in Fmoc-peptide gels for cell culture. Acta Biomater. 2009, 5, 934−943. (17) Debnath, S.; Shome, A.; Das, D.; Das, P. K. Hydrogelation through self-assembly of fmoc-peptide functionalized cationic amphiphiles: potent antibacterial agent. J. Phys. Chem. B 2010, 114, 4407−4415. (18) Williams, R. J.; Smith, A. M.; Collins, R.; Hodson, N.; Das, A. K.; Ulijn, R. V. Enzyme-assisted self-assembly under thermodynamic control. Nat. Nanotechnol. 2009, 4, 19−24. (19) Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins, R. F.; Turner, M. L.; Saiani, A.; Ulijn, R. V. Fmoc-diphenylalanine self assembles to a hydrogel via a novel architecture based on pi-pi interlocked beta-sheets. Adv. Mater. 2008, 20, 37−38. (20) Castelletto, V.; Cheng, G.; Greenland, B. W.; Hamley, I. W.; Harris, P. J. F. Tuning the self-assembly of the bioactive dipeptide lcarnosine by incorporation of a bulky aromatic substituent. Langmuir 2011, 27, 2980−2988.

tetramer model (Figure 3c). This hints at the notion that the antiparallel arrangement is in fact the most accurate depiction of the supramolecular structure. In any case, this work mainly indicates a more cautious interpretation of infrared results of short peptides will be required in the future but does not necessarily alter the conclusions of the parallel,26 antiparallel,19 or polyproline II models27 proposed for specific cases. Ultimately, further work and complementary techniques, like vibrational circular dichroism36,48,49 or multidimensional IR,5,50,51 will be required to establish if the antiparallel conformation is indeed the prominent one in our case.

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CONCLUSIONS In summary, we have demonstrated that for self-assembling Fmoc-containing peptides the presence of two separate peaks in the amide I region of the IR is not indicative of antiparallel βsheets. Instead, both experimental and computational results show that it is the presence of the carbamate moiety that is responsible for the 1690 cm−1 peak observed in these systems rather than a ν∥ component. A similar peak pattern can thus be expected for, for example, carboxybenzyl (Cbz) or naphthalene (Nap)-protected peptides. Although the calculated peak patterns and intensities qualitatively agree with experimental results, currently our DFT-based computational models are insufficient in size to allow us to categorically distinguish between different β-sheet arrangements. As a consequence, neither parallel nor antiparallel β-sheets can be ruled out as potential supramolecular structures on the basis of infrared spectroscopy alone. Regardless, it is clear that our computed infrared bands are sensitive toward the chemical structure and stacking arrangement of these molecules and are thus a useful diagnostic tool to determine different structural features. Work on these systems is currently ongoing to further refine the details that this approach can reveal.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and analysis of Fmc-AA, additional stacking conformations, stacking energy calculations, and additional vibrational frequency data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (R.V.U.); [email protected]. uk (T.T.). Author Contributions §

S.F., P.W.J.M.F., and I.R.S. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.T.H. thanks the EPSRC, UK, and European Research Council for funding. R.V.U. thanks the Leverhulme Trust (Leadership Award) and EPSRC (Advanced Fellowship). T.T. thanks the EPSRC and the Glasgow Centre for Physical Organic Chemistry for funding.

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REFERENCES

(1) Barth, A.; Zscherp, C. What vibrations tell us about proteins. Q. Rev. Biophys. 2002, 35, 369−430. E

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