Article pubs.acs.org/JPCB
FT-IR Spectroscopy and Density Functional Theory Calculations of 13 C Isotopologues of the Helical Peptide Z‑Aib6‑OtBu Timothy Zeko, Steven F. Hannigan,† Timothy Jacisin, Matthew J. Guberman-Pfeffer, Eric R. Falcone,‡ Melissa J. Guildford,§ Christopher Szabo, Kathryn E. Cole,∥ Jessica Placido,⊥ Erin Daly, and Matthew A. Kubasik* Department of Chemistry and Biochemistry, Fairfield University, Fairfield, Connecticut 06824, United States ABSTRACT: Isotope-edited FT-IR spectroscopy is a combined synthetic and spectroscopic method used to characterize local (e.g., residue-level) vibrational environments of biomolecules. We have prepared the 310 helical peptide Z-Aib6-OtBu and seven 13C-enriched analogues that vary only in the number and position(s) of 13 CO isotopic enrichment. FT-IR spectra of these eight peptides solvated in the nonpolar aprotic solvent dichloromethane have been collected and compared to frequency, intensity, and normal mode results of DFT calculations. Single 13C enrichment of amide functional groups tends to localize amide I vibrational eigenmodes, providing residue-specific information regarding the local environment (e.g., hydrogen bonding or solvent exposure) of the peptide bond. Double 13C enrichment of Z-Aib6-OtBu allows for examination of interamide coupling between two labeled amide functional groups, providing experimental evidence of interamide coupling in the context of 310 helical structure. Although the calculated and observed interamide couplings of Z-Aib6-OtBu are a few cm−1 and less, the eight peptides exhibit distinct infrared spectra, revealing details of interamide coupling and residue level vibrational environments.
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INTRODUCTION Amide functional groups of proteins and peptides provide absorption bands in vibrational spectra that are diagnostic of secondary structure.1−6 Beyond providing merely a unique “fingerprint”, the amide I band contains information regarding the helix, sheet, and coil structural content for these biopolymers. The spectral details of the amide I band (e.g., band position and shape) emerge from interamide coupling that is sensitive to interamide conformational geometry. Developing a quantitative understanding of the spectral features of the amide I band requires good computational methods and sound experimental models to disentangle the confounding effects of spectral congestion, hydration, and solvation. Good models will lead to an understanding of the geometric and electronic details of interamide coupling that is at the heart of the sensitivity of the amide I band to peptide conformation. Site-specific isotopic enrichment of amide carbonyl carbons with carbon-13 provides a means for extracting residue-level information from otherwise congested amide I spectral bands: enrichment with carbon-13 increases the reduced mass of amide I vibrational modes and lowers the frequency of absorption by ∼40 cm−1. In favorable cases, infrared features for enriched sites are observed at frequencies outside of the broad amide I envelope, and residue-level information may be obtained.3 Thermally induced changes of local regions of secondary structure have been monitored in polypeptides by employing a single7−10 or two (or more)11−27 carbon-13 labels. Conformation-dependent interamide coupling of carbon-13 enriched sites provides a spectroscopic signature for the melting © 2013 American Chemical Society
of local secondary structural geometries with increasing temperature and have been employed to examine the local thermal stability of α-helices,9−13 amyloid fibrils,14−19 helixturn-helix motif,20 and β-hairpin peptides.21−27 The design and quantitative interpretation of such experiments depend upon successful theoretical and experimental understanding of interamide coupling. The infrared spectra of model systems containing multiple 13CO oscillators, such as α-helical, alanine-rich peptides,9 and peptides forming antiparallel β-sheets28 are rarely fully interpretable by inspection. Instead, theoretical and computational models are required to explain features such as anomalous intensities and dispersion of normal-mode frequencies. Krimm and Bandekar found that force field parameters alone were insufficient to reproduce spectral dispersion observed of the infrared spectra of model systems known to adopt sheets and helices.6 They invoked transition dipole coupling (TDC) as a supplemental mechanism for interamide coupling. Torii and Tasumi reproduced the features of amide I region infrared spectra of a series of proteins using a “floating dipole model”, which employed only TDC.29 However, application of TDC models requires good estimates for unperturbed (i.e., diagonal) force constants, which may not be quantitatively available.30 Indeed, Torii and Tasumi invoked slight variations in amide I force constants belonging to β-sheet and α-helical secondary structures to bring their simulations into better agreement with experiment. In the past decade, Received: September 3, 2013 Revised: December 13, 2013 Published: December 13, 2013 58
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amide (−C(O)NH−) groups. In Z-Aibn-OtBu, the amide groups are scaffolded in place between the terminal carbamate and ester functional groups and give rise to n − 1 amide I eigenmodes/eigenfrequencies. The infrared bands associated with CO stretching of the terminal carbonyl groups (carbamate and ester) appear at higher frequency than the amide I envelope and provide a convenient standard for normalizing infrared spectra. Computationally, these short peptides are small enough to allow calculation of frequencies, eigenmodes, and oscillator strengths via modern DFT methods. The peptide of the current work, Z-Aib6-OtBu, was first characterized by Toniolo et al. nearly three decades ago.40 It has been shown to adopt a 310 helical structure by NMR,40 IR,40 and X-ray45 studies. In addition to the peptide 6, we have prepared seven additional isotopologues (Table 1) containing one or two 13C-enriched amide functional groups. FT-IR spectroscopic analysis and corresponding DFT calculations of the amide I region of these isotopologues are presented below.
quantum chemical methods (e.g., Hartree−Fock and DFT) have been used by a number of groups to make predictions of the influence of secondary structural influence on the frequencies and normal modes of short peptides.30−38 A combined spectroscopic (IR, VCD, Raman) and computational (DFT) study of interamide coupling employing short 310 helical peptide isotopologues has recently been published by Keiderling and co-workers. 39 They used the strongly helicogenic residue of α-aminoisobutyric acid (Aib) to promote helical structure in short peptides of six to eight residues in length, generating a series of isotopologues by placing zero, one or two carbon-13 enriched alanine residues within the sequence -Aib-Ala-Aib-Ala-Ala-Aib-. Their work examined interamide coupling in a short helical system amenable to computational modeling by DFT methods.39 The present study is a report of linear infrared spectra of the homo-oligomer Z-Aib6-OtBu (Figure 1), seven isotopically
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MATERIALS AND METHODS Peptide Synthesis. The synthetic methods of Toniolo et al. were used to prepare peptide hexamers.40 13C-labeled Aib was prepared using 13C KCN (98% 13C, Cambridge Isotope Laboratories) as a starting material and the methods of Hammer and co-workers for synthesis of an α,α-dialkylated amino acid analogue, and employing the Z group instead of Fmoc.47 13C-enriched Z-Aib-OH was then incorporated into oligomers identically to unlabeled material. C-terminal activation of Aib dimer intermediates, both labeled and unlabeled, was via oxazolones prepared with acetic anhydride. Peptides were purified using C18 reverse phase HPLC (Shimadzu). The preparation of the peptides and isotopic substitution were confirmed via LC/MS (Emeryville Pharmaceutical Services). FT-IR Spectroscopy. Linear FT-IR spectra were collected on a Bruker Tensor 27 spectrometer at 4 cm−1 resolution using Blackman-Harris 3-term apodization. The sample compartment was purged using a Balston FT-IR purge gas generator. Samples were held in a Pike Technologies demountable liquid cell with CaF2 windows and a 50 μm Teflon spacer. Maximum absorbance of the amide I band was kept below 0.08 for all samples. Dichloromethane solvent was stored over molecular sieves (4A) prior to use. Linear infrared spectra were fit to a sum of pseudo-Voigt line shapes of the form
Figure 1. Chemical structure of Peptide 6, showing 310 helical hydrogen bonding pattern (dotted lines) and enumerating CO bonds. 0, carbamate carbonyl; 1−5, Amide carbonyl groups; 6, ester carbonyl.
Table 1. Labeling Pattern and Notation of Isotopologues of Z-Aib6-OtBu Used in the Current Work notation
sequence
6 6.1 6.2 6.3 6.4 6.1.2 6.1.3 6.1.4
Z-Aib-Aib-Aib-Aib-Aib-Aib-OtBu Z-Aib*-Aib-Aib-Aib-Aib-Aib-OtBu Z-Aib-Aib*-Aib-Aib-Aib-Aib-OtBu Z-Aib-Aib- Aib*-Aib-Aib-Aib-OtBu Z-Aib-Aib-Aib-Aib*-Aib-Aib-OtBu Z-Aib*-Aib*-Aib-Aib-Aib-Aib-OtBu Z-Aib*-Aib-Aib*-Aib-Aib-Aib-OtBu Z-Aib*-Aib-Aib-Aib*-Aib-Aib-OtBu Aib* = 13C enrichment at carbonyl carbon.
⎡ 2 fwhm 1 + (1 − m) a ⎢m ⎢⎣ π [fwhm 2 + 4(v − vo)2 ] fwhm
varied versions (Table 1), and their corresponding DFT calculations. Homo-oligomers of Aib, Z-Aibn-OtBu, are favorable scaffolds for spectroscopic and computational studies of interamide coupling. Evidence from NMR,40 IR,40 2D-IR,41 and X-ray diffraction42−46 all confirm a reliable 310 helical structure for Z-Aibn-OtBu, even at lengths as short as n = 6. As compared to the α-helix, the 310 helix has a tighter pitch (3.0 residues/turn vs 3.6 for an α-helix) and an i → i + 3 hydrogen bonding pattern (instead of the i → i + 4 hydrogen bonding pattern of the α-helix). These oligomers dissolve in solvents of modest polarity (e.g., chloroform and dichloromethane), which reduces inhomogeneous broadening of infrared spectral features. The peptide Z-Aibn-OtBu contains n + 1 carbonyl-based functional groups: an N-terminal carbamate moiety (−OC( O)NH−), a C-terminal ester group (−C(O)O−), and n − 1
⎛ ⎛ v − vo ⎞2 ⎞⎤ 4ln 2 ⎟ ⎟⎥ + c exp⎜ − 4ln 2⎜ 0 ⎝ fwhm ⎠ ⎠⎥⎦ π ⎝
This line shape is a sum of Lorentzian and Gaussian functions with common peak position (νo), a conserved integrated area (a), conserved width (full width at half-maximum, fwhm), and decimal fraction of Lorentzian content (m). The Gaussian contribution is a pragmatic treatment of inhomogeneous line broadening. The peptide in this study, Z-Aib6-OtBu, offers five amide I modes. Two pseudo-Voigt components appeared to offer a qualitatively acceptable fit to the broad 12C amide I envelope. Additional fitting components, while connected to the physical reality of >2 amide I modes, did not qualitatively improve the 59
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fits or offer fitting parameters of increased quality. This strategy of using a minimal number of fitting parameters is consistent with that used in ref 39, where in some cases only one band was used to fit bands that originating from more than one vibrational mode. The 13C-based spectral features were fit with one pseudoVoigt line shape for each expected 13C amide I mode; that is, 13 C features of singly labeled peptides employed one pseudoVoigt line shape for the 13C band, while 13C spectral features of doubly labeled peptides were fit with two. DFT Calculations. Quantum chemical calculations were performed using Gaussian0948 running on a Ubuntu Linux computer. Typically, either 8 or 16 processors were used. DFT calculations employed PW91/6-31G(d) model chemistry. Torsional angles of the peptide were restrained to canonical φ, ψ angles. Two slightly different models for 310 helices were employed, either φ, ψ = −60, −30, or φ, ψ = −57, −30. Output files from Gaussian 09 were visualized using the open-source molecular visualization project Jmol.49,50 Jmol scripts, written in-house, analyzed and processed the atomic displacement vectors associated with individual eigenmodes. For each amide I eigenmode, the CO bond with the largest absolute displacement was identified. The projection of the displacement vectors of all of the other CO bonds onto the dominant CO vibration allowed their assignment as either “in-phase” or “out-of phase.”
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RESULTS AND DISCUSSION Quantum Chemical Calculations of Z-Aib6-OtBu as a 310 Helix. DFT calculations were performed constraining the peptides to φ = −60°, ψ = −30°, as in the work of Keiderling and co-workers.33,39 The peptide Z-Aib6-OtBu contains seven carbonyl oscillators, leading to seven vibrational eigenmodes that will be dominated by oscillations of CO bonds. Of these seven eigenmodes, five are amide I modes. The remaining two are carbamate and ester modes dominated by CO stretches of the N- and C-terminal groups, respectively. Peptide 6: Eigenfrequencies. DFT calculations provide frequencies of vibration (eigenvalues), normal modes (eigenmodes) and integrated spectral intensities. Figure 2 shows DFT results for the seven CO modes of peptide 6. Because Amide-I modes are known to be dominated by CO stretching, Figure 2 characterizes the eigenmodes of peptide 6 with bar graphs comparing the relative magnitudes of CO bond stretches of each CO bond in the peptide. In-phase/ out-of-phase oscillation of each CO bond is indicated by positive/negative bars, respectively. The structure of peptide 6 at the top of the figure aligns the CO bonds of peptide 6 with bars indicating the magnitude of their displacements. Analogous bar graphs have been used previously to illustrate the calculated three amide I normal modes of a tripeptide30 and the 28 amide I modes of glucagon.6,51 The dispersion of amide I eigenfrequencies is just under 30 cm−1. This dispersion can be attributed to factors such as position within the peptide (influence of helix macrodipole), peptide bond nonplanarity, and hydrogen-bonding to the peptide unit.2 For Z-Aib6-OtBu in a 310 helical conformation, the five amide units exhibit three different intramolecular hydrogen bonding conditions (Figure 1): (i) intramolecular hydrogen bonding to CO, only (CO of Aib1); (ii) intramolecular hydrogen bonding to both the oxygen of CO and the amide hydrogen (amide groups of CO of Aib2 and Aib3); and (iii) hydrogen bonding to the amide hydrogen of the
Figure 2. Carbonyl region eigenfrequencies (cm−1) and CO eigenmode characteristics of peptide 6. Eigenfrequencies are indexed in order of increasing calculated frequency, ν1−ν7. Value in parentheses is the calculated integrated absorption strength (KM/ mol). Bar graphs indicate relative CO bond displacements of each mode. Positive/negative bars indicate in-phase/out-of-phase stretching, respectively. Bars indicating the relative CO bond displacements are aligned beneath CO bonds of Z-Aib6-OtBu shown at the top of the figure. Black bars: relative displacements of amide group CO. Gray bars: displacement of CO carbamate and ester terminal groups.
amide unit, only (amide groups of CO of Aib4 and Aib5). Hydrogen bonding to an amide functional group is expected to lower its vibrational frequency. Indeed, the lowest calculated eigenfrequencies, ν1 and ν2, are modes dominated by displacements of CO bonds of Aib2 and Aib3, which are doubly hydrogen-bonded amide functional groups. These two eigenfrequencies belong to modes featuring strong interamide 60
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Figure 3. Carbonyl region eigenfrequencies (cm−1) and eigenmodes for eight isotopologues of Z-Aib6-OtBu. Displacements of 13CO bonds are shown in red. Displacements of carbamate CO and ester CO bonds are in gray. Positive/negative bars indicate in-phase/out-of-phase stretching, respectively. For all isotopologues, the mode assignments (ν1, ν2, and so forth) remain associated with the modes of CO displacement identified for the unlabeled peptide 6, so that for most isotopologues, ν1 is no longer the mode of lowest energy.
displaces the CO of Aib3 more than any other CO bond. This eigenmode contains significant out-of-phase coupling with the CO bond of Aib2. Interamide coupling depends upon the relative orientation of the oscillators (e.g., off-diagonal, TDC matrix element) and the similarity of the unperturbed vibrational frequencies (e.g., diagonal matrix element).2 The strong coupling of these two oscillators is due not only to their status as near-neighbors but also due to the closeness of their uncoupled, intrinsic frequencies. Much smaller contributions to the lowest-energy ν1 eigenmode arise from oscillations of the remaining carbonyl bonds in the peptide. The eigenmode of the next highest frequency, ν2, again shows strong coupling of the carbonyl oscillators of Aib3 and Aib2 in an in-phase manner. The strong, in-phase coupling of the two carbonyl groups is expected to enhance the oscillator
coupling between CO2 and CO3 (see below). The next three higher energy eigenfrequencies of amide functional groups singly hydrogen bonded are grouped within ∼12 cm−1 of each other and alternate in order of increasing energy as donor/acceptor/donor. The highest energy CO modes are calculated to be the carbamate and ester modes of the N- and C-terminal protecting groups, respectively. The bar graphs of the figure indicate that these eigenmodes are highly localized. Peptide 6: Delocalization of Eigenmodes. The bar graphs of Figure 2 clearly show that virtually all amide I eigenmodes are delocalized across several CO oscillators. This is necessarily the case for amide I modes, given the sensitivity of the amide I band to peptide conformation. The lowest frequency eigenmode for the unlabeled peptide 6, ν1, 61
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strength (i.e., lead to a more intense absorption) of ν2 as compared to ν1. (Figure 2 shows the calculated integrated absorption strengths of the modes in parentheses.) Additionally, mode ν2 contains enhanced contribution from in-phase oscillation of CO5. The oscillation of CO5 dominates the next higher-energy eigenmode, ν3. The eigenmode of ν3 is dominated by CO stretching of Aib5. The stretching of this carbonyl couples with modest outof-phase stretches of the carbonyl bonds of Aib3, Aib2, and Aib4. The net effect is a small integrated absorption strength for ν3 as compared to the other eigenmodes. The remaining amide I eigenfrequencies, ν4 and ν5 are dominated by CO stretching of Aib1 and Aib4, respectively, modestly delocalized among other amide-group CO stretches of the peptide. Isotopologues of Z-Aib6-OtBu: Eigenfrequencies and Eigenmodes. Figure 3 shows the DFT-based eigenfrequencies and eigenmodes for all isotopologues employed in this study. Figure 3 maintains the mode assignments (ν1, ν2, and so forth) of Figure 2; modes are assigned by the dominant CO displacements identified in the unlabeled peptide 6. Results for the unlabeled peptide 6 are in the first column for ease of comparison to results of Figure 2. Singly 13C-Enriched Isotopomers. Figure 3, column 6.1 displays the calculated eigenmode and eigenfrequency results for peptide 6.1 in which the carbonyl of Aib1 is enriched in carbon-13. DFT calculations predict that for isotopologue 6.1 the frequency of ν4 is shifted by ∼45 cm−1 to lower energy, as compared to ν4 of unlabeled pepitde 6 (column 6.1 vs column 6, row ν4 of Figure 3). Additionally, the bar graphs of Figure 3 show that the eigenmode of ν4 in isotopologue 6.1 is somewhat more localized as compared to peptide 6. The DFT calculations predict both a frequency shift and a reduction in intensity, since isotopically substituted and localized mode ν4 no longer benefits from in-phase coupling with the carbonyl stretches of Aib2 and Aib4. Figure 3, column 6.2 displays the calculated eigenmode and eigenfrequency results for isotopologue 6.2 in which the carbonyl of Aib2 is enriched in carbon-13. The eigenmode dominated by CO2 stretching is ν2. The frequency of this mode is also shifted to lower energy by ∼45 cm−1, and isotopic enrichment has the effect of localizing this mode to displacements of the 13CO. Since ν2 no longer benefits from in-phase coupling with CO3, the intensity of ν2 in peptide 6.2 is expected to diminish. Figure 3, column 6.3, shows DFT results for isotopologue 6.3. The isotopic shift experienced by ν1 is predicted to be ∼40 cm−1. However, the localization of this mode in peptide 6.3 is predicted to increase its intensity as compared to this mode in the unlabeled peptide, since out-of-phase interamide coupling of ν1 in unlabeled peptide 6 reduces its oscillator strength. DFT results for isotopomer 6.4, are shown in Figure 3, column 6.4. Stretching of CO4 dominates the mode belonging to ν5. Isotopic enrichment of CO4 with carbon13 is calculated to lower the energy of ν5 by ∼43 cm−1. The eigenmode of 13C-enriched ν5 retains small couplings to adjacent amide carbonyl bonds. While 13C substitution at Aib4 changes the pattern of coupling, DFT calculations predict little change in integrated absorption strength for this mode. Calculated frequency shift for isotopically enriched amide I modes are 40−45 cm−1. The frequency ordering of 13Cenriched modes of the four isotopomers is ν1(13CO3) < ν2(13CO2) < ν4(13CO1) < ν5(13CO4). The localization of the 13CO amide I modes provides characterization of the
amide I environment at the level of individual residues: The two lowest energy amide I frequencies belong to amide functional groups that are hydrogen bonding donors and acceptors, and the next highest frequency is of an amide hydrogen bound to its carbonyl oxygen, only. The highest frequency is predicted to belong to the amide with hydrogen bonding from the amide hydrogen, only. For the singly enriched isotopomers, isotopic enrichment with 13CO is predicted to perturb eigenfrequencies, eigenmodes, and oscillator strengths even for modes in which an unlabeled CO is the dominant oscillator. This phenomenon is a result of the delocalized nature of the amide I modes. As an example, ν1 in unlabeled peptide 6 has a calculated (vacuum) frequency of 1685.8 cm−1 and stretching of CO3, coupled in an out-of-phase fashion with stretching of the CO2 bond, dominates this mode. For peptide 6.2, in which CO2 is enriched with 13C, ν1 no longer exhibits strong out-of-phase coupling between CO3 and 13CO2. The predicted result is a frequency shift of ∼+3 cm−1 and an increase in absorption intensity for ν1. These changes result by limiting the participation of the 13CO2 oscillator in ν1 by shifting the intrinsic frequency of the CO2 oscillator. Perturbations of the 12C amide I band of 25-residue alaninebased isotopomers have been observed experimentally previously.52 Doubly 13C-enriched Isotopomers. Characterizing the interaction of two amide oscillators enriched in carbon-13 is the goal of double-13C substitution. While single substitution tends to isolate eigenfrequencies (by shifting them to frequencies lower than the broad amide I envelope) and localize eigenmodes dominated by 13CO stretching, double substitution has the potential to explore the coupling of two carbon-13 enriched amide oscillators by limiting interactions with other modes originating on amide functional groups containing 12C. When coupled, two 13CO oscillators produce two modes, νip and νop, characteristic of the in-phase and outof-phase eigenmodes, respectively. Our doubly enriched peptide isotopologue series can be characterized by zero (6.1.2), one (6.1.3), or two (6.1.4) unlabeled residues acting as “spacers” between carbon-13 enriched residues. Similar spacing schemes for doubly labeled peptides have been employed by other groups.9,39 The last three columns of Figure 3 show DFT results of eigenfrequencies and eigenmodes for the isotopologues 6.1.2, 6.1.3, and 6.1.4. DFT calculations show results similar to what has been previously reported for these spacing schemes. Adjacent 13C-enriched residues (6.1.2) couple strongly with νip (in-phase coupled frequency) of higher energy than that of νop (out-of-phase coupled frequency). 13C-enriched residues spaced by a single residue (6.1.3) show νip of lower energy than that of νop. In our calculations, residues spaced by two residues show virtually no coupling at all. (Compare rows ν4 and ν5 of 6.1, 6.4, and 6.1.4.) The most interesting comparisons are between 13CO dominated modes of singly labeled isotopologues with the corresponding modes of doubly labeled isotopologues. For example, mode ν2 of 6.2 is localized stretch of 13CO2, and mode ν4 of 6.1 is a localized mode of 13CO1. The frequencies of the ν2 and ν4 modes of doubly enriched isotopologue 6.1.2 are perturbed by interamide coupling between the amides containing 13CO1 and 13CO2. The figure shows that ν2 of 6.1.2 is ∼1.1 cm−1 lower in energy than ν2 of 6.2 and ν4 of 6.1.2 is ∼1.9 cm−1 higher in energy than ν4 of 6.1. 62
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Figure 4. (a−e) Experimental FT-IR spectra of the amide I region of isotopologues 6, 6.1, 6.2, 6.3, and 6.4 in dichloromethane solvent (black line). Constituent bands comprising the fits are shown in red; (f−j) DFT frequency and integrated intensity results (see Materials and Methods); simulated spectra result from application of a 20 cm−1 Gaussian line width.
cm−1, while 13C-localized amide I modes of 6.2 and 6.3 are ∼1650 and ∼1646 cm−1, respectively. As noted above, within a 310 helical secondary structure, the amide functional group containing CO1 is singly intramolecularly hydrogen bound to its carbonyl oxygen, whereas the amide groups containing C O2 and CO3 are intramolecular hydrogen bond donors and acceptors. The differences in intramolecular hydrogen bonding status between 13CO1 and 13CO2/13CO3 causes the intrinsic frequencies of these amide oscillators to diverge, limiting coupling. For the doubly labeled isotopologue 6.1.4, DFT calculations predict no change in the frequencies of the 13CO dominated modes ν4 and ν5, as compared to these same modes localized by single substitution. This is interesting because the corresponding localized 13C modes of 6.1 and 6.4 are predicted to be energetically separated by only ∼6 cm−1. For the 13CO
The doubly labeled isotopologue 6.1.3 contains 13C labels separated by a single residue. Furthermore, these two amide groups are brought into close proximity by a shared hydrogen bond. However, DFT calculations predict very small frequency perturbations of 13CO dominated modes ν1 and ν4, dominated by CO3 and CO1, respectively. The frequency of ν1 in 6.1.3 (small, in-phase coupling of 13CO3 and 13C O1) is ∼0.3 cm−1 lower in energy than the localized ν1 stretch of 6.3. The out-of-phase coupled mode ν4 of 6.1.3 (small outof-phase coupling of 13CO3 and 13CO1) shows a higher frequency of ∼0.7 cm−1 as compared to ν4 of 6.1. The very small effects predicted for these two pairs of doubly enriched isotopologues are due in part to the energetic mismatch of the corresponding localized 13C stretches predicted for the singly enriched analogues. For example, ν4 (13CO1) of 6.1 is predicted to have a frequency of ∼1661 63
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Figure 5. (a−c) Experimental FT-IR spectra of the amide I region of isotopologues 6.1.2, 6.1.3, and 6.1.4 in dichloromethane solvent (black line). Constituent bands (see Materials and Methods) to the fits are shown in red; (d−f) DFT frequency and integrated intensity results (see Materials and Methods) for 6.1.2, 6.1.3, and 6.1.4. Simulated spectra result from application of a 20 cm−1 Gaussian line width.
Table 2. DFT Calculated and Observed 13C Amide I Mode Maxima
oscillators of 6.1.4, geometric factors (e.g., distance) most likely precludes coupling. Linear FT-IR Spectroscopy. The Z-Aib6-OtBu isotopologues of this study contain the same seven carbonyl-containing functional groups: one N-terminal carbamate, one C-terminal ester, and five intervening amide groups. All seven of these carbonyl functional groups contribute spectral features in the carbonyl region of the infrared spectrum shown in Figures 4 and 5. The isotope-edited spectra of carbon-13 enriched isotopologues exhibit distinct features associated with carbon13 isotopic enrichment. FT-IR Spectra of Unlabeled and Single 13C Isotopologues. Figure 4a shows the carbonyl region of the FT-IR spectrum of the unlabeled peptide 6 solvated in dichloromethane. The carbamate and ester functional groups provide an infrared absorption feature at higher energy than the broad amide I band of peptide 6. This ester/carbamate band has been used to normalize the spectra. The intense amide I envelope is an asymmetric feature containing five overlapping amide I bands. The amide I envelope was fit to the sum of two pseudoVoigt functions (see Methods). No attempt was made to retrieve individual components of the five amide I eigenmodes that comprise the amide I band of peptide 6. The maximum absorption of the amide I band of peptide 6 is listed in Table 2. Figure 4f shows the FT-IR spectrum of peptide 6 simulated from the DFT results presented above. As expected, the in vacuo frequencies calculated by DFT methods are higher than observed frequencies.39 The singly enriched isotopologues (6.1, 6.2, 6.3, and 6.4) each show an amide I band shifted to lower energy due to the carbon-13 enrichment, as expected by simple reduced mass arguments. Additionally, as shown in the eigenmode plots
13
peptide 6 6.1 6.2 6.3 6.4 6.1.2 6.1.3 6.1.4 a
C amide I frequencies (cm−1)
DFT calculation 1661 1650 1646 1667 1649a 1646b 1661
1663b 1662a 1667
fit to data 1633.3 1625.8 1621.7 1634.0 1624.5 1621.3 1628.2
1636.4 1634.0 1635.2
Out-of-phase. bIn-phase.
(Figure 3), carbon-13 enrichment has the effect of localizing the eigenmode. Therefore the observed frequencies of these singly labeled isotopologues characterize the local environment of individual amide functional groups within the helix. Figure 4b shows the carbonyl region of the infrared spectrum of peptide 6.1. The isotope-shifted feature of the amide I mode originating from 13CO of Aib1 is a distinct band at lower frequency than the unlabeled amide I features. The individual pseudo-Voigt lineshapes that comprise the fit are shown. No attempt was made to resolve individual components of the broad 12C-based amide I band. 13C amide I peak positions obtained from spectral fits are in Table 2. Figure 4 shows the FT-IR spectra for 6.2, 6.3, and 6.4 in panels (d−f), respectively, along with the fits to the spectra along with the constituent pseudo-Voigt lineshapes. The qualitative match between the shapes of the calculated and observed spectra is very good. DFT calculations predict 64
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atoms, lowering observed amide I frequencies for amide functional groups not intramolecularly hydrogen-bound. FT-IR Spectra of Double 13C-labeled Isotopologues. Doubly 13C-enriched isotopologues provide two 13CO amide functional groups with the potential to interact through TDC and other mechanisms (e.g., mechanical coupling of near neighbors). Isotopologue 6.1.2 has near-neighbor carbon-13 enrichment, while the enriched amides of 6.1.3 are, within the context of a 310 helix, brought to close proximity via a direct hydrogen bond. (The 13C-enriched amide oscillators of 6.1.4 would be directly hydrogen bound if the peptide were to adopt an α-helix.) When coupled, two 13CO oscillators produce two modes, νip and νop, characteristic of the in-phase and outof-phase eigenmodes, respectively. The geometry of the 310 helix correlates the CO bond vectors. Each CO bond has a large positive projection along the helical axis; the CO bonds all point toward the Cterminus. Therefore, in-phase coupling of two different CO stretches will enhance the change of the dipole moment μ along the vibrational mode coordinate, (∂μ/∂Q)0, leading to a more intense band. Conversely, out-of-phase coupling of two CO bond stretches will diminish (∂μ/∂Q)0, reducing the oscillator strength, and observed intensity, of out-of-phase-coupled modes. Figure 5a−c shows experimental amide I region infrared spectra for three doubly 13C-enriched isotopologues 6.1.2, 6.1.3, and 6.1.4. Fits and DFT results are also plotted in the figure. Results of fits are collected in Table 2. The spectrum of peptide 6.1.2 with two nearest-neighbor amide units enriched in 13C (13CO of Aib1 and Aib2) exhibits a 13C amide I band arising from two 13C-based modes. It shows a poorly resolved low frequency shoulder. We assign this low intensity shoulder to νop; out-of-phase coupling of two nearly parallel 13CO stretches will reduce (∂μ/∂Q)0, leading to a spectral feature of diminished intensity. Assigning this lowintensity shoulder to νop confirms νop < νip for this peptide, consistent with the quantum chemical calculations and the results of other workers.39 Peptide 6.1.3 contains 13CO labeled amide units originating on Aib1 and Aib3. A single unlabeled residue separates the two enriched residues. In addition, in a 310 helical conformation, these two amide groups are brought into close proximity by a mutual hydrogen bond. The 13C spectral feature of this isotopologue exhibits a clear shoulder at higher frequency. The higher energy shoulder may be reasonably assigned to νop, giving νop > νip for this isotopomer. DFT results shown in the figure corroborate this assignment. Peptide 6.1.4 contains 13CO labeled amide units originating on Aib1 and Aib4. Two unlabeled residues separate these labels. They do not share a hydrogen bond with one another: Within the 310 helical secondary structure, the amide group containing the CO of Aib1 is a hydrogen bond acceptor (only) and the amide group containing the CO of Aib4 is a hydrogen bond donor (only). The DFT calculations suggest that this isotopologue will have essentially no coupling; the eigenmode plot of Figure 3 indicates that 13CO eigenmodes of this isotopologue are highly localized. Although this 13C infrared spectral feature of 6.1.4 is comparatively narrow and intense, it resisted being fit to a single pseudo-Voigt line shape. The best fits to this feature contained a sum of two pseudo-Voigt lineshapes with central frequencies differing by ∼7 cm−1. This compares well with the predicted frequency difference of ∼6 cm−1 by DFT calculations,
that the singly labeled isotopologues will exhibit FT-IR spectra that are distinct from the unlabeled peptide 6 and distinct from each other. The ordering of the observed 13C amide I frequencies among these isotopologues is ν6.3 < ν6.2 < ν6.1 < ν6.4, consistent with the ordering predicted by the DFT calculations (Table 2). Within the canonical hydrogen bonding pattern of a 310 helix, the 13CO of 6.1 is a hydrogen-bonding acceptor (only) and the amide group containing the 13CO of 6.4 is a hydrogen-bonding donor (only). These amide I bands will therefore be observed at higher frequencies compared to the 13CO amide I bands of 6.2 and 6.3, whose amide groups are both intramolecular hydrogen bond donors and acceptors. Combined with a generic Gaussian line shape, the DFT calculations predict frequencies and mode intensities that reproduce both the 13C amide I bands and the shapes of the broad 12C amide I envelopes quite well. For 6.4, the simulated 13C amide I CO4 feature appears to be less well resolved than observed experimentally. Because of a lack of hydrogen bonding to its carbonyl oxygen, the amide I mode dominated by displacement of 13CO4 is indeed expected to exhibit a higher frequency than the other 13Clabeled amide groups. However, the calculations appear to overestimate this phenomenon. Slight variations of the peptide structure in the vicinity of the C-terminus (e.g., conformational excursions away from canonical 310 helical structure) may be the origin of this discrepancy (see below). The FT-IR spectra of 6.1, 6.2, 6.3, and 6.4 contrast with existing literature spectra of 13C-labeled helical peptides in several ways. First, the great majority of “isotope-edited” studies have been performed on alanine-rich peptides of 17−32 residues in length in aqueous solution.9,52−55 Most reported amide I spectra of isotopomeric peptides employ multiple 13C labels. Localized to segments of model peptides, the 13C amide I spectral features revealed differential thermal stabilities of different regions of these model peptides, with the C-terminus typically “melting” or “fraying” at temperatures lower than the midhelix regions.11,54,55 A few studies have included a single 13 C amide I oscillator in the context of a long (25 residue) helical peptide.9 In the work of Decatur and co-workers, a small 13 C amide I band is observable at 2 °C, but disappears into the broad 12C amide I band, containing 24 12C amide I modes, as the temperature is raised. Keiderling and co-workers have reported FT-IR, VCD, and Raman spectroscopic data and DFT calculations of isotopologues of short (6−8 residue) peptides containing the helicogenic residue Aib.39 Their spectra of isotopologues solvated in TFE/CH3Cl serve as a point of comparison for the spectra presented here (Aib homooligomer isotopologues solvated in MeCl2). The spectra of the singly labeled isotopomers of the current work show much greater definition of the single 13C amide I features than previously reported. The sharpness of the spectral features in the current work may be due to the high helical secondary structural integrity of Z-Aib6OtBu, a reduced number of amide I modes of Z-Aib6-OtBu as compared to literature peptides, or reduced solvent−induced broadening by MeCl2 as compared to the polar protic solvent used in other studies (e.g., TFE). Keiderling and co-workers imply about a 5 cm−1 increase in the frequency of the 13C amide I band when the carbonyl oxygen atom lacks an intramolecular hydrogen-bonding partner.39 We observe a 9− 12 cm−1 increase. The protic solvent TFE used in ref 39 may offer hydrogen bonding partners to exposed carbonyl oxygen 65
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Eigenfrequencies and eigenmodes were then calculated from the structures. Unfortunately, the DFT results originating from X-ray data at any of the three levels of constraint failed to match the experimental spectra. In general, the DFT results for X-raystructure based results showed a greater dispersion of frequencies than observed by experiment. In particular, the fundamental frequencies CO stretches of the C-terminal CO and of Aib4 were calculated as too high. This could have been due to the helix-sense “reversal” of the C-terminal residue, preserved by all three levels of constraint, starting from the Xray coordinates. Among the isotopologues studied, the DFT calculations for 6.4 with uniform ψ, φ angles of −60, −30 agree least well with experiment. The 13CO4 amide I band is predicted as too high, and the shape of the broad 12C amide I envelope, as simulated, differs even qualitatively from experiment. Perhaps some “fraying” of the C-terminus precludes better agreement between calculation and experiment for isotopologues that contain 13CO4. Instead of X-ray structures, previous workers have invoked “canonical” (ψ, φ) angles for the 310 helix.39 We performed calculations constraining (ψ, φ) values to (−60, −30) or (−57, −30) and, optionally, constraining ω to 180°. Small variations in ω are supported by the X-ray structure of Z-Aib6-OtBu that shows an average deviation of ω from 180° by an average of ∼4.5° in the Z-Aib6-OtBu crystal structure.45 The results from constraining ψ = −60, φ = −30, and optimizing ω provided slightly better comparisons to experiment than ψ = −57, φ = −30 and are plotted and discussed above. The ordering of eigenfrequencies/eigenmodes and interamide coupling patterns were qualitatively similar between the two alternative ψ, φ parameters, (−60, −30) or (−57, −30).
but it is a significantly larger difference in frequency than suggested by the corresponding experimental spectra of singly labeled isotopomers 6.1 and 6.4. As noted above, the DFT result for 6.4 is “off trend” in that it predicts a higher frequency (less well resolved from the 12CO amide I envelope) for the 13 CO feature for 6.4 than is observed. Perhaps a spectrum of higher resolution or S/N is needed to reveal greater details of the 13C-based spectral feature of 6.1.4. Apart from 6.1.4, the DFT calculations of the isotopologues supply a starting point for course spectral simulations (Figure 5d−f). In 6.1.2 and 6.1.3, interamide coupling is clearly evident from the intensity differences between νip and νop of the 13C features. Despite the significant influence on spectral intensities, coupling-induced frequency shifts are quite small (Table 2). Isotopic substitution appears to influence the 12C amide I envelope of the simulated spectra, as well. The simulated and observed amide I region spectra agree in broad features for individual isotopologues/isotopomers and easily distinguish isotopologues/isotopomers from one another. The direct observation of νip (intense 13C amide I band) and νop (shoulder of 13C amide I band) in the spectra of 6.1.2 and 6.1.3 is a unique feature of this study. In contrast to previously reported spectra, our spectra contain features that may be assigned by intensity arguments to in-phase and out-of-phase coupling between 13CO oscillators. This system may reveal in-phase and out-of-phase features due to high-conformational homogeneity (reduction of inhomogeneous broadening), an aprotic environment (reducing both inhomogeneous broadening/solvent hydrogen bonding effects), a small number of amide I modes (reducing spectral overlap) and, perhaps most importantly, the coupling of modes whose intrinsic frequencies are nondegenerate. As noted above, the 13CO1 oscillator is an intramolecularly hydrogen-bonding acceptor, only, while the amide functional groups containing 13CO2 and 13CO3 are doubly hydrogen bound, lowering the uncoupled, intrinsic frequencies of their amide I modes. The dispersion of the 13C modes belonging to 6.1 versus 6.2 or 6.3 allows for the subtle effects of coupling (intensity and frequency changes) to be examined without significant overlap between coupled modes (e.g., 13C modes of 6.1.2 and 6.1.3). In this system, spectral dispersion that is not so great as to limit mode coupling but sufficient enough to reveal νin and νop. 310 Helical Peptide Geometry. The sensitivity of amide I eigenfrequencies to peptide conformation implies that the quality of quantum chemical computational results will depend upon the fidelity of the input geometry. We addressed this issue by testing a number of alternative helical geometries for Z-Aib6OtBu. (The i → i + 3 hydrogen bonding pattern can be achieved through slight variations among (φ, ψ) angles.) Initially, the crystal structure of Z-Aib6-OtBu appeared to be an attractive starting point for the determination of energyminimized structures in vacuo. However, the crystal structures of the series Z-Aibn-OtBu commonly exhibit a interesting reversal of helix handedness for the terminal Aib residue.46,56 This is true for the crystal structure of peptide employed in this study (n = 6).45 It is not known whether this phenomenon persists in solution for Aib oligomers C-terminated with −OtBu or simply a packing effect within the crystal. In any case, we performed DFT-based energy minimizations of peptide structure beginning with the published X-ray coordinates using three different levels of constraints: (i) constraining ψ, φ, and ω backbone dihedral angles; (ii) constraining only ψ and φ backbone dihedral angles, and (iii) fully unconstrained.
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CONCLUSIONS We have presented an experimental and computational (DFT) study of interamide coupling in eight isotopologues of the 310 helical peptide Z-Aib6-OtBu. The experimental spectra of singly 13 C-enriched isotopologues in the aprotic solvent dichloromethane offer residue-level information of the effect of intramolecular hydrogen bonding on amide I frequencies. The experimental spectra of the doubly 13 C-enriched isotopologues offer clear, qualitative picture of near neighbor and next-near neighbor coupling and assignment of νip (inphase) and νop (out-of-phase coupling). For Z-Aib6-OtBu adopting a 310 helix, coupling between CO1/CO2 and C O1/CO3 is evident experimentally and computationally. However, coupling between CO1 and CO4 is more nuanced. Computationally, no coupling is observed. Experimentally, spectral overlap of the modes originating on these two carbonyl groups prevents resolving a clear picture. Since the intrinsic frequencies of CO1 and CO4 could be expected to be fairly close (only one intramolecular hydrogen bond to each amide), geometric factors (e.g., distance) likely limit coupling. The DFT results of modest computational cost compare well with the observed spectra, in terms of 13C-induced spectral shifts, the ordering of energies of the single 13C isotopologues and interamide couplings of the double 13C isotopologues. Despite the rather small changes in frequencies induced by coupling between amide groups, changes in vibrational modes (e.g., integrated band intensities) provide characteristic lineshapes that qualitatively reproduce experimental spectra. Two-dimensional IR studies of these isotopologues are planned. Additionally, experiments of longer (e.g., n = 8) and 66
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(6) Krimm, S.; Bandekar, J. Vibrational Spectroscopy and Conformation of Peptides, Polypeptides, and Proteins. Adv. Protein Chem. 1986, 38, 181−364. (7) Brewer, S. H.; Song, B.; Raleigh, D. P.; Dyer, R. B. Residue Specific Resolution of Protein Folding Dynamics using Isotope-Edited Infrared Temperature Jump Spectroscopy. Biochemistry 2007, 46, 3279−3285. (8) Fang, C.; Hochstrasser, R. M. Two-Dimensional Infrared Spectra of the 13C=18O Isotopomers of Alanine Residues in an α-Helix. J. Phys. Chem. B 2005, 109, 18652−18663. (9) Barber-Armstrong, W.; Donaldson, T.; Wijesooriya, H.; Silva, R. A. G. D.; Decatur, S. M. Empirical Relationships between IsotopeEdited IR Spectra and Helix Geometry in Model Peptides. J. Am. Chem. Soc. 2004, 126, 2339−2345. (10) Huang, R.; Kubelka, J.; Barber-Armstrong, W.; Silva, R. A. G. D.; Decatur, S. M.; Keiderling, T. A. Nature of Vibrational Coupling in Helical Peptides: An Isotopic Labeling Study. J. Am. Chem. Soc. 2004, 126, 2346−2354. (11) Silva, R. A. G. D.; Kubelka, J.; Bour, P.; Decatur, S. M.; Keiderling, T. A. Site-Specific Conformational Determination in Thermal Unfolding Studies of Helical Peptides using Vibrational Circular Dichroism. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8318−8323. (12) Huang, C. Y.; Getahun, Z.; Zhu, Y. J.; Klemke, J. W.; DeGrado, W. F.; Gai, F. Helix Formation Via Conformation Diffusion Search. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2788−2793. (13) Ramajo, A. P.; Petty, S. A.; Starzyk, A.; Decatur, S. M.; Volk, M. The α-Helix Folds More Rapidly at the C-Terminus than at the NTerminus. J. Am. Chem. Soc. 2005, 127, 13784−13785. (14) Petty, S. A.; Decatur, S. M. Experimental Evidence for the Reorganization of β-Strands within Aggregates of the Aβ(16−22) Peptide. J. Am. Chem. Soc. 2005, 127, 13488−13489. (15) Petty, S. A.; Decatur, S. M. Intersheet Rearrangement of Polypeptides during Nucleation of β-Sheet Aggregates. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 14272−14277. (16) Kim, Y. S.; Liu, L.; Axelsen, P. H.; Hochstrasser, R. M. TwoDimensional Infrared Spectra of Isotopically Diluted Amyloid Fibrils from Aβ40. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 7720−7725. (17) Moran, S. D.; Decatur, S. M.; Zanni, M. T. Structural and Sequence Analysis of the Human γD-Crystallin Amyloid Fibril Core using 2D IR Spectroscopy, Segmental 13C Labeling, and Mass Spectrometry. J. Am. Chem. Soc. 2012, 134, 18410−18416. (18) Moran, S. D.; Woys, A. M.; Buchanan, L. E.; Bixby, E.; Decatur, S. M.; Zanni, M. T. Two-Dimensional IR Spectroscopy and Segmental 13 C Labeling Reveals the Domain Structure of Human γD-Crystallin Amyloid Fibrils. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 3329−3334. (19) Shim, S. H.; Gupta, R.; Ling, Y. L.; Strasfeld, D. B.; Raleigh, D. P.; Zanni, M. T. Two-Dimensional IR Spectroscopy and Isotope Labeling Defines the Pathway of Amyloid Formation with ResidueSpecific Resolution. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 6614− 6619. (20) Amunson, K. E.; Ackels, L.; Kubelka, J. Site-Specific Unfolding Thermodynamics of a Helix-Turn-Helix Protein. J. Am. Chem. Soc. 2008, 130, 8146−8147. (21) Hauser, K.; Krejtschi, C.; Huang, R.; Wu, L.; Keiderling, T. A. Site-Specific Relaxation Kinetics of a Tryptophan Zipper Hairpin Peptide using Temperature-Jump IR Spectroscopy and Isotopic Labeling. J. Am. Chem. Soc. 2008, 130, 2984−2992. (22) Setnicka, V.; Huang, R.; Thomas, C. L.; Etienne, M. A.; Kubelka, J.; Hammer, R. P.; Keiderling, T. A. IR Study of Cross-Strand Coupling in a β-Hairpin Peptide using Isotopic Labels. J. Am. Chem. Soc. 2005, 127, 4992−4993. (23) Huang, R.; Wu, L.; McElheny, D.; Bour, P.; Roy, A.; Keiderling, T. A. Cross-Strand Coupling and Site-Specific Unfolding Thermodynamics of a Trpzip β-Hairpin Peptide using C-13 Isotopic Labeling and IR Spectroscopy. J. Phys. Chem. B 2009, 113, 5661−5674. (24) Huang, R.; Setnicka, V.; Etienne, M. A.; Kim, J.; Kubelka, J.; Hammer, R. P.; Keiderling, T. A. Cross-Strand Coupling of a βHairpin Peptide Stabilized with an Aib-Gly Turn Studied using
C- and N-terminal group analog peptides and peptide isotopomers are in progress. These studies will examine coupling among 13C-labeled amides that are internal to helical peptide secondary structure.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: mkubasik@fairfield.edu. Fax: 203-254-4034. Present Addresses †
(S.F.H) Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, MA 02215. ‡ (E.F) Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269−3092 § (M.J.G.) Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School, 303 Belmont Street, Worcester, MA 01604 ∥ (K.E.C) Department of Molecular Biology and Chemistry, Christopher Newport University, 1 Avenue of the Arts, Newport News, VA 23606. ⊥ (J.P.) Department of Toxicology, St. John’s University, 8000 Utopia Parkway, Jamaica, NY 11439. Author Contributions
M.A.K. synthesized, purified and collected FT-IR of isotopologoues, performed and analyzed DFT calculations, and wrote the manuscript. T.Z. synthesized and purified peptides and collected FT-IR spectra. M.J.G.-P. performed DFT calculations. S.F.H. collected FT-IR spectra. T.Z., T.J., M.J.G., E.D., C.S., K.E.C., and J.P. synthesized labeled and unlabeled peptides and peptide fragments. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to acknowledge the administrative assistance of Ms. Lori Fahy and laboratory managerial assistance of Dr. Dorothy Sobczynski in support of this research. The research of this manuscript was supported in part by grants from the American Chemical Society’s Petroleum Research Fund (ACS-PRF #33928-GB4) and a Cottrell College Science Award from Research Corporation (CC5899).
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ABBREVIATIONS DFT, density functional theory; Aib, α-aminoisobutryic acid; Ala, L-alanine; Z, benzyloxycarbonyl; OtBu, tert-butoxy; FT-IR, Fourier transform infrared; νip, frequency of in-phase mode; νop, frequency of out-of-phase mode
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REFERENCES
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