Experimental and Theoretical Spectroscopic Study of 310-Helical

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Experimental and Theoretical Spectroscopic Study of 310-Helical Peptides Using Isotopic Labeling to Evaluate Vibrational Coupling Ahmed Lakhani,† Anjan Roy,† Matteo De Poli,‡ Marcelo Nakaema,† Fernando Formaggio,‡ Claudio Toniolo,‡ and Timothy A. Keiderling*,† † ‡

Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607-7061, United States Institute of Biomolecular Chemistry, Padova Unit, CNR, and Department of Chemistry, University of Padova, 35131 Padova, Italy

bS Supporting Information ABSTRACT: Coupling between the amide linkages in a peptide or protein is the key physical property that gives vibrational spectra and circular dichroism sensitivity to secondary structures. By use of 13 C isotopic labeling on individual and pairs of amide CdO groups, the amide I band for selected residues was effectively isolated in designed hexa- and octapeptides having dominant 310-helical conformations. The resultant frequency and intensity responses were measured with IR absorption, vibrational circular dichroism (VCD), and Raman spectroscopies and simulated with density functional theory (DFT) based computations. Band fitting the spectral components and correlating the results to the computed coupling between selected labeled positions were used to determine coupling constant signs and to estimate their magnitudes for specific sequences. The observed frequency and intensity patterns, and their variation between IR and VCD with label position in the sequence, follow the theoretical predictions to a large degree, but are complicated by end effects that alter the local force field (FF) for some residues in these short peptides. These FF variations were overestimated in the theoretical models which may be evidence of structural variations not included in the model. By analyzing the simulations with different coupling models, the coupling constants were determined to lie in a range (positive) þ35 cm1 for sequential residues (i,iþ1) and with (negative) 3 cm1 as an upper bound for alternate ones (i,iþ2). The sequential amide coupling for 310-helices is weaker than for R-helices but has the same sign and is larger than and oppositely signed as compared to 31-, or poly-(Pro)n type-II, helices.

’ INTRODUCTION Determination of peptide and protein secondary structure often relies on frequency shifts for characteristic vibrational transitions (using IR absorption or Raman spectroscopy) or on band shapes and intensity patterns (using circular dichroism, CD). In each case, the resultant spectral parameters are the consequences of coupling between sequential amide groups in the oligo- (or poly-) peptide chains. Since the transitions arising from different sites in the sequence overlap, these spectrally determined structural components are necessarily averages for the entire molecule and are not spatially or sequentially distinct. However, with vibrational spectra it is possible to gain site specificity through isotopic labeling.13 In particular, 13C substitution on the amide CdO results in a shift of the amide I mode to lower frequency by ∼40 cm1, which usually effects a resolution of the contribution of those labeled residues from that of the 12CdO groups (even larger shifts are possible with 13Cd18O labeling4). If two or more sites are labeled, the position of the observed 13CdO band is dependent on their mutual coupling as well as the basic shifts caused by isotopic substitution. This coupling is the fundamental interaction between amides that gives vibrational spectroscopy its sensitivity to secondary structure, and thus it has an intrinsic value that has been evaluated r 2011 American Chemical Society

both empirically for structure prediction as well as theoretically for model evaluation. We and several other groups have prepared a variety of smallto medium-length peptides which incorporate selected isotopic labels to enable vibrational spectra to follow localized structural changes.126 In some of our studies, comparison of experimental spectra with results of theoretical simulation using density functional theory (DFT) based computations for peptides with two labels incorporated on specific sites were undertaken to obtain a measure of the coupling constants.2,1416,27,28 Many previous isotopic labeling studies depended on IR absorption spectra of the amide I mode (primarily CdO stretch) and on simulation of such spectra for a peptide segment constrained to the structure being modeled. In general, the observed position of the coupled 13CdO band differed if two labels were placed adjacent or separated by one or more residues in the sequences, as was most clearly evident for R-helical peptides.2 Separating the labels reduces their coupling but can also change the sign of the coupling constant so that the more Received: January 11, 2011 Revised: March 19, 2011 Published: April 18, 2011 6252

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The Journal of Physical Chemistry B intense coupled component might be either higher or lower in frequency from the isotope shifted position of an isolated amide 13 CdO.2,14 The latter can be determined empirically by preparation of the same peptide sequence with just one labeled residue. For β-sheet structure, the behavior is more complex, yet still can be modeled with DFT-based theoretical methods.15,16,19,27,29 Despite being resolved from the 12CdO component, the 13 CdO contributions from two or more labeled residues to the amide I overlap each other in IR absorption or Raman spectra. Thus their splitting, and hence coupling constant, cannot be directly measured. However, due to the complementary natures of the IR absorption and Raman techniques, the resultant 13 CdO amide I contributions can have different intensity distributions, i.e. the in-phase coupling can be weaker or stronger than the out-of-phase coupling, for a given structure and technique. These differences can enable determination of at least a lower bound to the coupling constant, depending on the relative IR absorption and Raman intensities of the coupled components. This method is most useful for β-sheet-like structures. However, for some conformations, since the intensity patterns for the oppositely phased IR absorption and Raman components are similar, an empirical measure of the coupling constant cannot be obtained with just these spectra because the observed band has the same frequency position in both. For coupling amide I modes for two labeled 13CdO groups, the vibrational CD (VCD) will obviously correspond to coupled modes with the same frequency splitting, but in this case the two components will normally have oppositely signed intensities. When the observed frequencies of these VCD component bands are compared with the IR band position, the combined IR absorption and VCD spectra can provide a measure of another bound to the coupling constant. Alternatively, IR absorption, Raman and VCD spectra can be simulated using DFT methods and the resultant spectral patterns can be compared to those observed experimentally. Achieving agreement between simulated and experimental spectral patterns thus validates the sign and order of magnitude obtained for the computed coupling constant. Previously, we have studied vibrational coupling for conformationally stable peptide sequences in R-helical1,2 and β-sheet19,29 conformations by use of isotopic labeling and DFT modeling. These were extended by similar but alternately designed studies of β-hairpins.15,16,27,28 More recently, we investigated other helical forms, initially reporting analyses for poly-(Pro)n type-II (PPII) or 31-helices.14 In the present study, we report experimental and theoretical investigations for synthetic 310-helical peptides,30 which are models for the fourth most common secondary structure motif in proteins, that accounts for 10% of helical residues in protein structures.3133 The 310-helix is characterized by a tighter twist of the backbone as compared to that of the R-helix and its intramolecular CdO 3 3 3 H—N H-bonds are oriented at a slightly larger angle to the helical axis. This latter property results in less stability of the 310-helix motif as compared to the R-helix, which is evidenced primarily for longer peptides, particularly in H-bonding solvents.3235 While the ideal approach might seem to involve preparation of very long 310-helical peptides with two central residues labeled with 13CdO, such an approach does not work in practice, since stabilized uniform 310-helices cannot be obtained with longer sequences. For shorter structures, 310-helix structure formation is favored by incorporation of residues having dialkyl

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Table 1. Amino Acid Sequences and Labeling Patterns of the Peptides Used in This Study notation

peptide sequence

UH

iPrCO-Aib-Ala-Aib-Ala-Ala-Aib-NHiPr

A4H A5H

iPrCO-Aib-Ala-Aib-Alaa-Ala-Aib-NHiPr iPrCO-Aib-Ala-Aib-Ala-Alaa-Aib-NHiPr

A4A5H

iPrCO-Aib-Ala-Aib-Alaa-Alaa-Aib-NHiPr

A2A4H

iPrCO-Aib-Alaa-Aib-Alaa-Ala-Aib-NHiPr

A2A5H

iPrCO-Aib-Alaa-Aib-Ala-Alaa-Aib-NHiPr

A4O

Ac-Aib-Ala-Aib-Alaa-Ala-Aib-Aib-Aib-OMe

A5O

Ac-Aib-Ala-Aib-Ala-Alaa-Aib-Aib-Aib-OMe

A4A5O

Ac-Aib-Ala-Aib-Alaa-Alaa-Aib-Aib-Aib-OMe

a 13

C labeled on the amide CdO.

substitution on the R-carbon that restricts the rotational freedom of the backbone (φ,ψ) torsion angles.3638 Thus, incorporation of Aib (R-aminoisobutyric acid or CR,R-dimethylglycine) residues and restriction to peptides of limited lengths can favor 310-helix formation. For convenience of isotopic labeling, we prepared a series of mixed Aib/Ala-containing hexapeptide amides and an octapeptide ester. Previous studies from our laboratories indicated that a host Aib oligomer with incorporation of up to three guest protein residues in a terminally protected (or blocked) hexapeptide amide or octapeptide ester could yield a 310-helical structure if dissolved in a solvent like CDCl3.33,34,3941 Obviously use of such short lengths suggests questioning the conformational homogeneity, particularly at the ends, but obtaining a 310 structure is the critical first step in such a targetted coupling study and correction for the design shortcomings must come second. Recently 13C, 18O, and 15N isotopically labeled 310-helical peptides have also been targets of coherent 2D IR studies to observe crosspeaks between the amide I and amide II modes coupled through a CdO 3 3 3 H—N intramolecular H-bond.13 For the present study, IR absorption, VCD, and Raman spectra were obtained for several single- and double-labeled, terminally blocked peptides (Table 1), which all had the same basic sequence, -Aib-Ala-Aib-Ala-Ala-Aib-, and these experimental spectra are compared to their DFT simulations. The doublelabeled hexapeptides varied only in sequential position of the isotopic label (13CdO) on two of the Ala residues to yield three different compounds, designated as A4A5H, A2A4H, and A2A5H. In addition, an octapeptide, -Aib-Ala-Aib-Ala-Ala-AibAib-Aib-, was synthesized with double labels as A4A5O to avoid end effects on the Ala5 label position in the hexapeptides. Finally, the unlabeled UH and the single labeled A4H and A5H hexapeptides were prepared to provide spectral references for adjusting our DFT force field. A preliminary account of a limited part of this work was presented in a symposium publication.42

’ MATERIALS AND METHODS Peptide Synthesis and Characterization. Peptide synthesis was performed by solution methods, using the EDC, 1[3-(dimethylamino)propyl]-3-ethylcarbodiimide/HOBt, 1-hydroxy-1,2,3-benzotriazole,43 or, more efficiently, the EDC/ HOAt, 7-aza-1-hydroxy-1,2,3-benzotriazole,44 C-activation procedure. Coupling reactions were conducted in anhydrous CH2Cl2 in the presence of diisopropylethylamine. All purified peptides were characterized by melting point (where appropriate) and polarimetric determinations, solid-state IR absorption, 6253

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Figure 1. Ideal geometry of the 310-helical conformation of the hexapeptide UH used for DFT spectral simulations. The H-bonds (CdO 3 3 3 H—N) are indicated as red dashed lines.

and 1H NMR (see the Supporting Information). The final hexaand octapeptides were additionally checked by HPLC and mass spectrometry. For labeling, the commercial (Cambridge Isotopes, Andover, MA) R-amino acid H-(1-13C)L-Ala-OH was used. The peptide sequences studied and our notation for them are summarized in Table 1. The N-terminus of each hexapeptide was blocked with isopropylcarbonyl (iPrCO) and the C-terminus with isopropylamino (NHiPr). The octapeptide was blocked with acetyl (Ac) at the N-terminus and protected at the C-terminus as a methyl ester (OMe). For spectral measurements, all peptide samples were dissolved in 2,2,2-trifluoeoethanol (TFE) or in a TFE/CHCl3 (1:3 v/v) mixed solvent. Spectral Measurements. Electronic CD (ECD): Far-UV ECD spectra of peptides were measured using a Jasco J-810 spectropolarimeter for peptides dissolved in TFE (∼0.2 mg/mL) and placed in a 1-mm quartz cuvette. Eight scans were collected between 185 and 250 at 50 nm/min, with a 1-nm bandwidth and 2 s response time. These spectra were averaged and corrected with a baseline identically obtained for just the solvent in the same sample cell. FT-IR. Mid-IR absorption spectra were recorded on a Digilab FTS-60A FT-IR spectrophotometer equipped with a DTGS detector. The peptides were dissolved in TFE or in TFE/CHCl3 (1520 mg/mL), placed in a homemade demountable sample cell composed of two CaF2 windows separated by a (50 μ) Teflon spacer. Absorbances of ∼0.35 to ∼0.60 in amide I and ∼0.30 to ∼0.50 in amide II were normally obtained. Spectra reported were an average of 128 scans at 4 cm1 resolution, referenced to identical scans of an empty sample beam and corrected by subtraction with a background (solvent) spectrum measured under similar conditions. VCD. All VCD spectra were measured using a homemade dispersive instrument separately described in detail.45 Briefly, it consists of a 0.3 m monochromator (Acton Research, SpectraPro 2300i), C-Rod source, 57 kHz CaF2 modulator (Hinds International), and a narrow band liquid-nitrogen-cooled MCT detector (Infrared Assoc.). Data are processed with independent digitization of transmission and modulation intensity and ratioed in a control computer programmed in LabView. The same samples for IR absorption were again used for VCD measurements but with 12 cm1 spectral resolution. Although individual scans had a very good S/N ratio, typically we averaged spectra from eight scans over the amide I and II regions, ∼17501450 cm1

to obtain stable, well-reproduced results for these peptides. Baseline correction was obtained by identical scans of a separate cell containing only TFE or TFE/CHCl3 and then subtracting the resulting spectrum from the sample spectrum. Raman. Raman measurements were obtained for peptides at ∼100 mg/mL in TFE or TFE/CHCl3 solutions. Our homemade Raman spectrometer used a 785 nm (380 mW) excitation laser (Innovative Photonic Solutions), passing through a laser-line filter (Semrock), and focused with 5-cm focal length lens into a quartz sample cell. [Use of an Ar ion laser, 514 nm, resulted in substantial background fluorescence with these samples.] The 90° collection geometry includes an edge filter (Semrock) to block the laser lines before focusing the scattered light on the entrance slit of a 0.64 m, f/5.4 monochromator (Jobin-Yvon) equipped with a back-illuminated CCD detector (Newton, Andor Technology). A good S/N ratio was obtained after 6 h of acquisition for each peptide at this concentration. Raman spectra were background corrected in the amide I region by subtracting a linear baseline from the sample spectra. Computation Method. Molecular models of the hexa- and octapeptides were created using standard 310-helix (φ,ψ) torsion angles. All the DFT calculations were performed using Gaussian 03 on a Linux based machine, usually with four 64-bit processors and shared memory (16 GB).46 The peptide structure was constrained to an ideal 310-helix conformation, (φ,ψ) equal to (60°,30°), for the results reported here, but was optimized for all other coordinates. Alternate geometries were also tested. The sequence was blocked with Ac- on the N-terminus and methylamino (NHMe) on the C-terminus. The force field (FF), atomic polar (APT for IR intensity), polarizability (for Raman), and axial tensors (AAT for VCD intensity) were then computed at the 6-31G*/BPW91 level. Computations for the peptide in vacuo and in a simulated solvent using a polarized continuum model (PCM) were undertaken. Simulation of spectral bandshapes involved summing Gaussian lineshapes assigned to each computed mode frequency and scaled to the intensity for the spectrum being computed.47,48 Variations with isotopic labeling patterns were simulated by changing masses and rediagonalizing the FF.

’ RESULTS ECD and VCD Results to Establish 310-Helical Peptide Conformations. Figure 1 shows a representation of the 6254


The Journal of Physical Chemistry B 310-helix geometry of UH used to compute the FF and intensity parameters for the mixed Ala/Aib hexapeptides. Even for this ideal structure, the CdO at the Ala5 (and Aib6) position has no intermolecular H-bond since the peptide has no amide —NH at the corresponding i þ 3 positions (discussed below in terms of end effects). This was the motivation for preparing a blocked octapeptide with two additional Aib residues on the C-terminus. To experimentally determine the presence of a 310-helical structure, we used a combined analysis of ECD49,50 and VCD spectroscopic results.33,34,39,51,52 The ratio between the intensities at 222 and 205 nm in the ECD spectrum, designated as the R value, has been used to distinguish 310- and R-helices.33,50,5254 R values of 0.30.4 favor a 310-helix structure while R ∼ 1.0 are typically found for R-helices. Although somewhat dependent on R-alkylation and on environment, these distinctions have been shown to be qualitatively useful. The far-UV ECD spectra of the labeled A4H, A2A4H, and A4A5O measured in TFE (Figure 2a) all exhibit a significant negative band at 204 nm and a weak negative shoulder at ∼222 nm, with an R value of 0.350.40, consistent with a largely 310-helical conformation. Similar R values were obtained for the other hexapeptides (data not shown). The VCD spectrum of a 310-helical conformation gives rise to a positive couplet (positive to lower energy) for the amide I band and a stronger, relatively sharp negative amide II band, while the same sign patterns are observed for an R-helix, but its amide I VCD is dominant and amide II is broad and weak.2,33,34,40,52,55 Again, these patterns are somewhat sensitive to the degree of CRalkylation and have small frequency shifts in different solvents. The intensity ratio between the amide I couplet and amide II negative VCD is an important characteristic for distinguishing 310- and R-helices when coupled with their detailed band shapes as secondary evidence. The VCD spectra measured in TFE exhibit this pattern, but it is sharper and more intense in CDCl3 (mixed with TFE to enhance peptide solubility), as shown in Figure 2b for the amide I and II spectra of the unlabeled (UH) and the labeled A4A5H and A4A5O peptides where both VCD and IR were normalized to the area of the amide I absorption band. Due to normalization, the intensities should be independent of length to first order, which is approximately seen. The relative bandshapes for these peptides are consistent with that of a 310-helix, although the amide II (ΔA/A ∼ 15 105) appears weaker, and amide I appears somewhat stronger and more negatively biased (ΔA/A ∼ 10 to þ5 105) than expected, which is probably a result of inclusion of Ala and reduction of Aib residues in the sequence.34 The spectrum in the amide I region is more complex and weaker for the A4A5H and A4A5O peptides due to labeling, as discussed below, but the dominant 310-helical patterns remain quite evident. The comparison of the A4A5H and A4A5O indicates that adding two Aib residues maintains the 310-helix conformation. Despite A4A5O being longer, which could destabilize the 310-helix, its VCD does not indicate an Rhelical component and fits previously reported patterns for an octapeptide with a higher fraction of Aib residues.39,40 Similar relative amide I and II VCD lineshapes were observed for all other peptides investigated in this work. Unlabeled and Single Labeled Peptides Used to Establish Isotopic Effects (Diagonal FF). The IR absorption spectrum of the unlabeled (UH) peptide in the amide I region (Figure 3a, central panel) consists of a single band with slightly asymmetrical line shape to the lower energy side. This band can be fit to two Gaussian components, a main one at ∼1660 cm1 and a small

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Figure 2. (a) Experimental far-UV ECD spectra of A4H (solid line), A2A4H (dashed line), and A4A5O (dotteddashed line). The spectra were measured in TFE from 185 to 250 nm and corrected for prepared concentration and residue number. (b) IR absorption and (c) VCD spectra in the amide I and amide II band regions of peptides UH (solid line), A4A5H (dashed line), and A4A5O (dotted-dashed line) measured in TFE/CHCl3 (1:3 v/v) and normalized to the areas of the amide I band (scale arbitrary).

feature at ∼1630 cm1 which both probably result from exciton coupled 12CdO local stretching modes that make up the amide I band. The observed unlabeled hexapeptide amide I VCD spectra (Figure 3a, upper panel) has a positive couplet shape, going positive then negative (lower to higher frequency), indicative of right-handed helical formation. This couplet could be fit to three Gaussian components, but details of the VCD fits have proven less reliable than for the IR absorption and Raman fits, and thus will not be discussed further. For the single labeled A4H and A5H peptides, an additional 13 CdO band appears in the IR absorption spectrum (Figure 3b,c) 6255



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Figure 3. Experimental VCD (upper panel), IR absorption (middle panel), and Raman (bottom panel) spectra of (a) UH, (b) A4, and (c) A5. The spectra, measured in TFE/CHCl3 (1:3 v/v), only show the amide I region. The Gaussian components (red) and overall fit (green) in the IR absorption and Raman spectra are indicated with dashed lines.

as a shoulder on the lower energy side of the amide I band. The A5H 13CdO mode was fit to a band component at higher energy (∼1626 cm1) than that for A4H (∼1621 cm1). The difference arises because the Ala5 CdO group does not have a H-bond to another amide group, which results in that local mode having a higher energy than if it were intramolecularly H-bonded, as for Ala4. The significance of there not being a H-bond (end effect) at Ala5 will be addressed further with regard to comparisons of double labeled hexa- and octapeptides. The measured A4H and A5H VCD, shown in Figures 3b and 3c (upper panel), have somewhat reduced intensities and bandshapes that resemble those of UH, since no identifiable VCD features are seen due to the single 13CdO label. This lack of 13 CdO VCD is a consequence of its reduced coupling with other (12CdO) vibrations and offers graphic evidence of the importance of coupling in providing structural sensitivity to the spectral response. The lower VCD intensity for A4H and A5H is due to its having one less coupled 12CdO oscillator and somewhat broader spectra. Broadening is probably due to the isotope shift causing a break in the exciton coupling which disperses the intensity over more modes instead of concentrating it in one or two coupled modes and is regularly evident in simulations of IR spectra for isotopically labeled peptides.48 The Raman spectra (lower panels in Figure 3) reflect the IR absorption result, showing just a weak shoulder to low frequency on the main Raman amide I band. In each case, the IR absorption

band (12CdO) maximum is at ∼1660 cm1, while that of the Raman band is at ∼1658 cm1. This difference is due to the relative intensity distribution among the exciton split amide I modes, as confirmed by the simulations. The calculated IR absorption and VCD spectra for the idealized 310-helix geometry qualitatively agree with the UH experimental spectra as shown for the amide I band in Figure 4a. The main differences are that the calculated amide I frequency is too high (4045 cm1), as is normal for DFT FF, and the 12 CdO—13CdO separation is too large for A4H in comparison to the experimentally observed patterns. Compared to the experimental results, these calculated isotope shifts for A4H and A5H differ by too much. The A4H simulation has qualitative agreement with experiment, but the isotope shift is too large, while for the A5H (Figure 4c) the computed 13CdO mode is predicted at too high a frequency, due to the lack of H-bond, and does not result in a distinguishable shoulder on the 12CdO amide I band. This difference between the Ala4 and Ala5 CdO frequencies has two sources, the FF overestimation of both the end effect for Ala5 (frequency too high) and the isotope effect for Ala4 (frequency too low). The source of this error for Ala4 is tied to the partial disruption of the excitonic coupling as seen by the increased dispersion of the 12CdO amide I mode intensities (and consequent loss of overall peak intensity) for A4H. Nonetheless, these effects (mostly H-bond formation differences plus neglect of helical fraying) clearly lead to a nondegeneracy in the 6256



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Figure 4. Simulated IR absorption (bottom panel) and VCD (upper panel) spectra of (a) UH, (b) A4, and (c) A5, as represented by the band shape envelopes and vertical lines indicating the D and R values of the normal modes. Simulated IR absorption of UH is fitted with one Gaussian component, whereas those of A4H and A5H are fitted with two Gaussians indicated with dashed lines (red), and their sum is the green envelope in IR and the blue in VCD.

two local oscillators (Ala4 and Ala5) which can be seen experimentally (but less) and must be accounted for in order to analyze the coupling between pairs of CdO groups on different sites. This phenomenon provided a continuing challenge in interpreting the theoretical simulation analyses of the experimental spectra. Comparative IR and VCD of Double Labeled Hexa- and Octapeptides to Show Site-Specific Coupling. Sequential Labels. We first consider labeled peptides that have sequential (nearest neighbor) sites labeled. Two peptides were prepared, A4A5H, consistent with the other hexapeptide sequences studied, and A4A5O to minimize the impact of end effects on the labeled sites in A4A5H by placing the Ala4 and Ala5 positions well within the 310-helical fold so they were internally H-bonded to amides further in the sequence. The experimental amide I IR absorption, VCD, and Raman spectra for the double isotope labeled A4A5H hexapeptide and A4A5O octapeptide are shown in Figure 5a and b (middle panel). For both, the 13CdO component in the IR absorption and Raman spectra appears as a pronounced shoulder on the 12CdO amide I band. The amide I IR and Raman are qualitatively equivalent, and an additional band is observed for A4A5O at ∼1725 cm1, due to the ester (OMe) protecting group at the C-terminus. The 13CdO sideband is shifted to lower frequency in A4A5O, presumably due to its two coupled modes having more comparable H-bond formation (Ala5 is not H-bonded in the hexapeptide), reducing the overlap of 12CdO and 13CdO bands for A4A5O. It also has reduced relative intensity, since there are proportionately fewer 13 CdO oscillators and less spectral overlap between the 12CdO and 13CdO contributions in A4A5O. The corresponding VCD spectra for both A4A5H and A4A5O have a positive couplet shape in both the 13CdO and 12 CdO amide I bands, with positive lobe to the lower frequency side of the corresponding IR absorption band, but due to overlap and to the negative bias of the dominant 12CdO couplet, the

positive contributions are diminished. The isotope shifted A4A5H VCD appears to be relatively more intense, which is partly due to the enhanced fraction of 12CdO and its increased VCD intensity in A4A5O, plus some effects of the nondegeneracy of the Ala4 and Ala5 13CdO oscillators in the hexapeptide. The ratio of ΔA/A for the 13CdO bands in each peptide is comparable. These VCD sign patterns and relative frequency positions to the IR reflect our results for sequential labels in R-helical peptides2 and are qualitatively predicted by our computational models; however, the intensities are quite different (see section following). Separated Labels. To complement the study of adjacent or sequential coupling, we prepared two samples A2A4H and A2A5H with labels separated by one and two residues, respectively, with the expectation of seeing reduced coupling between the labeled sites, as expected from basic physical interaction models and as has been observed for other peptide conformations.2,3,1416,19 Experimentally, separating the labels has a relatively minor impact on the IR absorption and Raman spectra, which again are qualitatively similar in the amide I (Figure 6, middle and lower panels), but has much greater effect on the VCD band shape, as seen in (Figure 6, upper panels). In the IR and Raman spectra, a 13CdO band resolved from the 12 CdO band is evident in A2A4H and A2A5H but is weaker in intensity than seen above for A4A5H. The 13CdO component for A2A4H is broader than for A2A5H and slightly higher in wavenumber. In the VCD the two separated labels for these 310-helical peptides give rise to very different patterns. The A2A4H peptide (Figure 6a, top) has a quite broad negative component and a weak positive one that appears to the high frequency side of the 13 CdO IR absorption band. By contrast, the A2A5H VCD (Figure 6b, top) has a narrower negative 12CdO VCD band but with a weak negative shoulder to low frequency between it and a resolved positive 13CdO band on the low frequency side of the corresponding IR absorption shoulder. 6257



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Figure 5. Experimental VCD (upper panel), IR absorption (middle panel), and Raman (bottom panel) spectra of (a) A4A5H and (b) A4A5O, measured and fit to Gaussian components as in Figure 3. A4A5O has an ester CdO stretch, due to the C-terminal (OMe) cap, near 1725 cm1.

Finally, contrasting the two sets of doubly labeled peptides, the A4A5O 12CdO band is sharper than that for A4A5H, suggesting a more uniform conformation, and better overlaps the A2A4H absorbance, suggesting less disruption due to end effects. Both A2A4H and A4A5O have both 13CdO modes intramolecularly H-bonded, thereby eliminating distortion and overlap on the low wavenumber side of the 12CdO band by the Ala5 13CdO, which is a problem for A4A5H and A2A5H. All the doubly labeled peptides have broader IR than the A4, singly labeled variant. Computational Modeling of the IR and VCD Spectra of the Coupled Residues. Due to the anomalous structure (lack of H-bond) for the Ala5 position 13CdO in the hexapeptide, the single mode frequencies were not well-represented in the modeling of A5H, as was shown above. Even when a solvent correction for CDCl3 using the PCM model was used, the results were fundamentally the same. The modes all shift down by ∼25 cm1, but their relative separations and intensities remain unchanged. Consequently, the computed spectra for the double labeled A4A5H and A2A5H were expected to have difficulty reproducing the experimental results, so that their simulated bandshapes are most useful when viewed in terms of differences from predictions for peptides with single labels and each other. By contrast, the A2A4H and A4A5O peptides are labeled so as to minimize the end effects, and those provide useful references for the A4A5H and A2A5H

experimental spectra. Consequently the spectral simulations for all the peptides are best discussed together, rather than individually with the spectral data. The amide I IR absorption bands for each of the double labeled peptides are predicted to have a resolved 13CdO sideband, but the relative intensities match the experiment only for the A2A4H and A4A5O, which have internally H-bonded labeled CdO groups. The simulated 13 CdO IR bands for A4A5H and A2A5H are too weak because only one mode (computed at ∼1652 cm1) contributes to it. The other 13CdO mode appears as the lowest frequency component (∼1674 cm1) of the apparent exciton broadened 12 CdO band (the center of which fits to a broadened Gaussian at ∼1698 cm1). The frequencies for the computed 13CdO modes and the fitted intensity bandshapes (as shown in Figures 7 and 8) are listed in Table 2. [It is important to distinguish between mode and band profile positions, as the observed spectra reflect the latter and their shifts from the modes for 13CdO are indicative of our ability to derive coupling constants from those spectra. The computed modes are all high by ∼50 cm1 due to lack of correction for solvent effects and to normal DFT errors. These can be partially corrected by using the PCM solvation model for CDCl3, which results in similar, but slightly reduced mode separations, as also summarized in Table2. The line widths used in the figures for the contributions of individual components are 6258



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Figure 6. Experimental VCD (upper panel), IR absorption (middle panel), and Raman (bottom panel) spectra of (a) A2A4H and (b) A2A5H, measured and fit to Gaussian components as in Figure 3.

a compromise between mimicking overall absorbance shapes and preserving the fluctuations in the VCD band profile, despite the overestimated Ala4 and Ala5 frequency differences.] As determined from our simulations, the IR absorption and Raman amide I spectra (Raman not shown) are predicted to have very similar intensity patterns, making their measurement only confirmatory for 310-helices, in contrast to the complementary isotope shifts found for other structures such as β-sheets or the 31- or PPII-helices.14 The simulated A4A5O VCD spectrum has a clear positive couplet 12CdO amide I, followed by a positive 13CdO couplet (Figure 7b, upper panel), which is in qualitative agreement with the experimental results (Figure 5b), but the computed 13CdO component is too intense. Attempts to measure the spectrum with higher resolution (not shown) gave essentially the same spectral result as seen in Figure 5. The simulated VCD spectra (Figure 7a) for A4A5H also qualitatively agree with the experimental spectra (Figure 5a), but now the 13CdO contribution to the VCD is too weak. These disparities in intensities and analyses in terms of coupling constants are topics in the Discussion.

’ DISCUSSION Coupling Analysis. To better analyze and compare these overlapping contributions between different labeling patterns, all the IR absorption spectra were fit to a minimal number of Gaussian band components to give the same type of fit pattern to each spectrum, experimental and simulated, even if use of more components might have reduced the overall fitting error. From this point of view, the IR and Raman fits are quite acceptable and virtually the same in terms of components (see Figures 3, 5, and 6), a systematic variation develops with isotope substitution pattern and the comparison of experiment and theory are qualitatively correct (Figures 7 and 8). The largest errors in the calculations for single label species regarding the 12 CdO to 13CdO differences are for A4H, calculated too large 13 ( CdO low), and A5H, too small (13CdO high). The fit bands for the computed 13CdO IR absorption components in A4A5H and A2A5H are ∼68 cm1 higher in frequency than for A2A4H, which is in qualitative agreement with the A4A5H fitted experimental 13CdO band being ∼6 cm1 higher than that for A2A4H, as summarized in Table 3. The A4A5O and A4A5H 6259



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Figure 7. Simulated IR absorption (bottom panel) and VCD (upper panel) spectra of (a) A4A5H and (b) A4A5O, plotted and fit as in Figure 4. An additional peak, to compensate for the C-terminal (OMe) ester group, is indicated as a blue dashed line.

experimental and theoretical fit band positions have roughly the same 12CdO to 13CdO separation (∼42 cm1), but the source of this apparent agreement is more complex due to the impact of the high-frequency Ala5 mode. The quality of the simulated VCD patterns in both A4A5H and A4A5O, as shown in Figure 7 (upper panel), appear to be surprisingly good given the impact of end effects on the mode mixing for A4A5H. Presumably this is because the primary criterion determining the 13CdO VCD pattern is the sign of the coupling constant between the two labeled residues. This sign (positive) is correctly predicted (which is easiest to see in the VCD pattern) and is the same in both of these sequentially labeled peptides, although the splitting is larger for A4A5H due to the end effect. Thus, the experimental IR absorption frequency is higher (overlaps the 12CdO more), and the relative VCD contribution is larger for A4A5H as well, due to effects of the local Ala5 and Ala4 mode (diagonal FF) differences. The simulations appear to overestimate the impact of internal H-bonding, resulting in too large a difference in diagonal FF for Ala4 and Ala5, as seen in the A4H and A5H mode positions in Table 2. However, there is another contribution, since the Ala4 substitution in A4H causes disruption in the amide I exciton coupling (evidenced as increased intensity dispersion, see Figure 4) that is less evident in A5H, which complicates the analysis. The impact of this error on the position of the fitted Gaussian components in A4A5H and A4A5O is small for the IR, due to convolution of multiple overlapping broadened

components. The computed 13CdO VCD for the octapeptide is larger than for the hexapeptide, despite the smaller separation of the Ala4 and Ala5 modes in A4A5O, in contrast to the complex mode mixing in A4A5H. This affects the overlap of positive and negative 12CdO VCD contributions in the middle of the amide I band, resulting in more cancellation between 12CdO VCD components and a relatively intense 13CdO contribution. The overestimation of the difference of the diagonal FF for Ala4 and Ala5 makes the sequential coupling (A4A5) analysis more complex than for the alternate positions as in A2A4H. For the simulated A2A4H peptide spectra, as shown in Figure 8a, the more intense, in-phase mode is computed at a lower frequency (1648 cm1) while the weaker, out-of-phase mode is at a higher frequency (1654 cm1), with a mode splitting of 6 cm1 (Table 2). The corresponding VCD components are opposite in sign (negative to low frequency) and roughly equal in magnitude, confirming the negative sign for the coupling. If we assume that the uncoupled, Ala2 and Ala4 amide I local modes were degenerate, then the coupling constant required to yield this splitting would be 3 cm1. This assumption is supported by the relatively large difference in Raman intensities computed for the two bands (not shown), implying strong mixing, and by the near degenerate computed frequencies obtained with test calculations isolating the Ala4 (1652 cm1) and Ala2 (1651 cm1) 13 CdO modes. (If this calculation were corrected for a nondegeneracy of 1 cm1, the coupling would be ∼2.9 cm1, see the Supporting Information, Figure S1.) The virtue of this model is 6260



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Figure 8. Simulated IR absorption (bottom panel) and VCD (upper panel) spectra of (a) A2A4H and (b) A2A5H, plotted and fit as in Figure 4.

Table 2. DFT Computed Frequency Values (Normal Modes) for Ideal 310-Helices and Fits to IR Simulations 13

notation

a

12

C mode frequency (cm1)

vacuum

PCMCHCl3

1652

1627

1651

A5H A4O

1674 1657

1651

1675 1655

A5O

1652

A4A5H

1675

1652

A2A5H

1674

1627

1651

a

1654

1652

a

1659

A2A4H

experimental IR notation

1652

b

b

C/13C Gaussian fit peak positions (cm1)

intensity fit

A4H

A4A5O

Table 3. Experimental and Theoretical Band Fitted Values for the Peptides Investigated

1659 1658

1648

1629

1625

1650

1651

1652

1627

1655

b

Asymmetric mode. Symmetric mode.

in its agreement with the IR experimental spectra (Figures 6a and 8a and Table 3). The computed VCD also agrees with experiment, being dominated by a broad negative to high frequency and with the positive 13CdO component higher in frequency than the corresponding IR absorption band and a weak negative VCD to lower frequency in both experiment and simulation.

12

C

theoretical Raman

13

C

12

C

13

IR C

1659

12

C

13

C

UH

1660

A4H

1660

1622

1659

1622

1698 1700

1651

A5H

1659

1627

1659

1624

1697

1675

A4A5H

1660

1623

1661

1626

1697

1659

A2A4H

1661

1618

1662

1618

1700

1650

A2A5H

1659

1618

1662

1620

1697

1655

A4A5O

1665

1626

1663

1629

1700

1658

Higher resolution might better define the 13CdO contributions to VCD, but the signals are predicted to be small and our preliminary higher resolution experimental VCD tests did not yield added band shape variation. Thus, this comparison to the experimental spectra supports the validity of the computed 6261


The Journal of Physical Chemistry B e3 cm1 coupling constant, which is quite comparable in sign and magnitude to the i f i þ 2 coupling constant derived for R-helices.2 Such a simple approximation of directly using the mode splitting to determine the coupling constant for A4A5H can not work, due to the end effects on the Ala5 13CdO group. However, for A4A5O, the 13CdO components are resolved from the 12CdO band, and the two coupled modes have quite different IR and Raman intensities, with the more intense inphase mode to higher frequency, indicating strong mixing. The corresponding VCD components are opposite in sign, negative to high frequency, and of equivalent magnitude. All of these 13CdO spectral predictions are strongly correlated to the experimental observations and suggest coupling of near degenerate modes, much as seen for A2A4H above, except that the coupling for A4A5O (and A4A5H) is positive. This A4A5O pattern agrees with that of A4A5H, but is there less evident (IR intensities are similar and VCD are more different, both due to nondegeneracy), and both fit the patterns seen for sequential labeling in R-helices due to the positive coupling.2 By parallel reasoning as for A2A4H, we can analyze the A4A5O simulation to determine the adjacent residue amide I coupling by use of the degenerate coupled oscillator assumption. The computed splitting of ∼8 cm1 (Table 2) would then correspond to twice the coupling giving a coupling constant of ∼4 cm1, which is smaller than that found for R-helices (∼7 cm1).2 The computed frequencies for the Ala4 and Ala5 amide I modes (for simulations of labeled octapeptide species) are not degenerate (Table 2, Figure S1, Supporting Information). This deviation is probably an overestimate, but if taken into account, the coupling would be reduced to ∼3.1 cm1. Similarly a coupling constant of ∼5 cm1 can be obtained from a nondegenerate analysis of the A4A5H spectrum (see the Supporting Information), suggesting a range of 35 cm1 for our coupling constant determination for sequential labels in a 310-helix. Thus the more tightly wound 310-helix maintains the same sign pattern as the R-helix in terms of coupling patterns for the i, i þ 1 and i, i þ 2 interactions, but the magnitude of the closer, adjacent sequential interaction is decreased. For the i, i þ 3 interaction, the sign pattern is less clear, and the A2A5H data unfortunately do not provide sufficient data to sort it out. The coupling sign for A2A5H appears to be opposite that of A2A4H, as can be derived from the relative frequency patterns of the experimental IR and VCD; however, in this case the calculated patterns are unclear and do not support a certain analysis of a positive sign for the coupling. Due to the small coupling constant magnitude combined with large nondegenerate FF contributions (Ala5), it is not possible to derive an experimental or theoretical value for A2A5H. Despite the successes for the A2A4H spectrum and the A4A5O and A4A5H 13CdO modes, in terms of coupling, there are difficulties in the intensity distributions for the latter. While the computed IR of A4A5O matches the experiment well, its computed VCD is too intense for the 13 CdO band (ΔA/A is roughly twice the experimental value) and too weak for the 12CdO band (here ΔA/A is about half the experimental value). Modified FF Analyses. The methods above use simple twomode coupling models for the DFT determined modes and gain applicability by the match of experimental and simulated bandshapes for IR and VCD (and Raman by extension). Our DFT FF calculations take into account all the modes of the molecule and

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are done at a relatively high level. Also, they should reflect the impact of variation along the sequence. However, as noted above, the Ala4 and Ala5 diagonal FF values are computed too low and high, respectively, which can dramatically impact comparison to experiment for A4A5H. To further test this point, we need to compare experimental and theoretical results in more detail. As previously noted, the theoretical simulations are all in qualitative agreement with the amide I experimental IR absorption and VCD (as well as the Raman) results. However, the A4A5H 13 CdO IR absorption (and Raman) intensity is underestimated while its VCD is in good agreement with the experiment. By contrast, the A4A5O 13CdO contribution is computed well for the IR absorption, but its VCD result is too large. [Note that the ester function resulting from the —OMe blocking group on the C-terminus has a band at ∼1725 cm1 experimentally, which, accounting for the expected DFT error, is well-predicted in the A4A5O simulation, but high as usual at ∼1757 cm1. This mode may impact the 12CdO mixing somewhat, as suggested by comparing detailed dipole and rotational strength dispersions for the simulations in Figure 7b.] This variation in intensity patterns shows a weakness in our FF determination, which may be a consequence of solvation being left out of the model or a consequence of using the ideal 310-helix structure (φ = 60o, ψ = 30°) in creating the model. The simplest correction is to account for solvation by use of a PCM model. As seen in the values listed in Table 2, columns 4 and 5, the inclusion of a dielectric corresponding to the CDCl3 solvent shifts to the frequencies by ∼25 cm1 but does not affect the relative splittings of the 13CdO bands or the shapes of the simulated amide I spectra (Figure S1, Supporting Information). [Other analyses have used or derived different torsional angle values (φ = 57o, ψ = 30°), but our PCM calculations with this geometry give frequency values that track with PCM results for the ideal structure (Figure S1, Supporting Information).13] Consequently, we have attempted to correct the local FF for the Ala4 and Ala5 positions in the hexapeptide. By reducing the Ala5 CdO force constant so that its amide I mode moved to position nearly degenerate with the Ala4, little change was seen in the IR absorption and VCD patterns. Both spectral simulations remain in qualitative agreement with the experiment, but the predicted 13CdO IR absorption intensity is still too low. One can alternatively increase the diagonal FF for Ala4, but this still fails to reproduce the two nearly equivalent bands seen in the experimental A4A5H IR absorption (and Raman) with a reasonable range of corrections in our computations. Alternatively, we modified the octapeptide FF to attempt to obtain a better model of the VCD intensity, since its IR absorption was acceptably accurate. For the A4A5O FF, the Ala4 and Ala5 13CdO modes are still not degenerate, with Ala4 being computed 5 cm1 higher than Ala5 (Table 2). This phenomenon impacts the splitting of the coupled 13CdO modes and their simulated VCD intensity (by reducing overlap). We could remove this nondegeneracy by FF modification, and the coupled labeled modes show strong mixing (symmetric mode dominating the IR intensity) for A4A5O with a splitting of >7 cm1 or a coupling of >3.5 cm1, in excellent agreement with the results following our degenerate and nondegenerate coupling analyses above. When Ala4 and Ala5 are made degenerate, the computed 13CdO VCD decreased somewhat but was still much more intense than that seen experimentally. This suggests that FF refinement can have an impact, but the FF changes we made are small compared to the probable impact of structure 6262


The Journal of Physical Chemistry B fluctuations on the dynamic peptide in solution. Accounting for such variation is beyond the scope of this work. Thus in both cases, the simple local FF correction is not the dominant source of intensity overestimation for the 13CdO A4A5H IR or A4A5O VCD. It can be noted that substitution of Ala4 vs Ala5 with 13CdO does impact the exciton coupling which effectively changes the local FF at those positions in the octapeptide. The effect of inserting one label is small, but it varies for different sequential positions in the hexapeptide (see Figure 4) which may explain why the A4H and A5H peptides differ so much in the simulations. At this level, we must conclude that the mismatch of IR absorption and VCD intensities in A4A5H and A4A5O is due to more fundamental aspects of the FF or to nonuniform distortion of the experimental geometry from the ideal one used for our simulations.

’ CONCLUSION We have evaluated the coupling between amide CdO groups on adjacent and separated residues in a series of 310-helical model peptides. Values determined are smaller than those found for R-helices for adjacent residues and about the same for separated ones. The sign patterns are the same for R- and 310-helices. The adjacent CdO groups have a positive coupling, yielding the same VCD sign pattern and a more intense IR component to higher energy (closer to the 12CdO band). The CdO groups separated by one residue have the opposite sign pattern. The computations support the observed isotopic shifts and splittings of coupled modes and can be analyzed to determine coupling constants, but the details seem dependent on minor changes in the FF or geometry which were not included in the modeling, suggesting treating our results as ranges. VCD for 310-helices is weaker than for the corresponding R-helices due to this weaker coupling. This reduced coupling appears to result from the different angle of the H-bonded CdO groups with respect to the helix axis in the 310-helices as compared to the R-helix H-bonding pattern. ’ ASSOCIATED CONTENT

bS

Supporting Information. Details of the peptide synthesis and characterizations are provided along with added computational results and coupling constant determinations for PCM corrected FF and for alternate geometries. This material is available free of charge via the Internet at http://pubs.acs.org .

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: (312) 996-0431.

’ ACKNOWLEDGMENT This work was supported at UIC by a grant from The National Science Foundation (CHE0718543). ’ REFERENCES (1) Silva, R. A. G. D.; Kubelka, J.; Decatur, S. M.; Bour, P.; Keiderling, T. A. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 8318. (2) Huang, R.; Kubelka, J.; Barber-Armstrong, W.; Silva, R. A. G. D.; Decatur, S. M.; Keiderling, T. A. J. Am. Chem. Soc. 2004, 126, 2346. (3) Decatur, S. M. Acc. Chem. Res. 2006, 39, 169. (4) Torres, J.; Kukol, A.; Goodman, J. N.; Arkin, I. T. Biopolymers 2001, 59, 396.

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Experimental and Theoretical Spectroscopic Study of 310-Helical

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