Azido Gauche Effect on the Backbone Conformation of β-Azidoalanine

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J. Phys. Chem. B 2010, 114, 13021–13029

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Azido Gauche Effect on the Backbone Conformation of β-Azidoalanine Peptides Kwang-Im Oh,† Woosung Kim,† Cheonik Joo,† Dong-Geun Yoo,‡ Hogyu Han,*,† Geum-Sook Hwang,‡ and Minhaeng Cho*,†,‡ Department of Chemistry, Korea UniVersity, Seoul 136-701, Korea, and Korea Basic Science Institute, Seoul 136-713, Korea ReceiVed: August 5, 2010

To study the azido gauche effect on the backbone conformation of β-azidoalanine (Aza) dipeptide (AAD, Ac-Aza-NHMe) and tripeptide (AAT, Ac-Aza-Aza-NH2), we used spectroscopic methods in combination with quantum chemistry calculations and molecular dynamics (MD) simulations. From the 1H NMR coupling constants and 1H,1H NOESY experimental data, we found that AAD in water mainly adopts a seven-membered cyclic (C7) rather than polyproline II (PII) backbone conformation and prefers the gauche- (g-) side-chain conformer. From the amide I IR absorption and circular dichroism (CD) spectra, the backbone conformation of AAD in water is found to deviate from PII but is rather close to C7. Thus, the backbone conformation of AAD differs from that of alanine dipeptide (AD, Ac-Ala-NHMe), which is mainly PII in water. The underlying origin of the backbone conformational difference between AAD and AD in water was elucidated by quantum chemistry calculations with density functional theory (DFT). It was found that the C7/g- conformer is the lowest energy structure of an isolated AAD. Here, the β-azido group forms intramolecular electrostatic interactions with two neighboring peptide bonds, which are facilitated by the azido gauche effect. Thus, the β-azido group appears to be responsible for directing the peptide backbone conformation toward the C7 structure. The quantum mechanical/molecular mechanical (QM/MM) MD simulations show that AAD in water adopts neither PII nor right-handed R-helix (RR) and prefers the g- conformer. Thus, the intramolecular electrostatic interactions between the β-azido group and two nearby peptide bonds are also found even in the aqueous solution structure of AAD. Consequently, the β-azido group appears to be an effective C7-conformationdirecting element, which may also be useful for tuning the structures of other amino acids and polypeptides. I. Introduction The “gauche effect” describes the apparent and anomalous preference for the gauche conformer over the anti conformer.1-7 This effect has been attributed primarily to σ-hyperconjugation, which describes the hyperconjugative electron donation of the bonding orbital (σ) into the antibonding orbital (σ*), consequently placing the best σ-donor bond anti to the best σ-acceptor bond. In the absence of strong interactions, this relatively weak gauche effect can have a large influence on the conformation of molecules. Such an effect is known for many molecules X-C-C-Y, where at least X or Y is an electron-withdrawing substituent. The gauche effect enables the creation of the conformation-controlled amino acids such as 4-fluoroproline and 4-azidoproline.1,4 These modified amino acids are also found to enhance the stability of collagen, allowing new biomaterials to be produced.2,3 However, the gauche effect is rarely obtained with other natural and artificially modified amino acids for controlling their conformation. Recently, we reported that β-azidoalanine can be a potentially useful IR probe of the local electrostatic environment in protein aggregates.8 β-Azidoalanine incorporated into Aβ(16-22) peptide of the Alzheimer’s disease amyloid β-protein at position Ala21 enabled the IR probing of the hydrophobic environment in peptide aggregates. Such IR probing with β-azidoalanine was made possible by a few notable characteristic features of the * To whom correspondence should be addressed. E-mail: mcho@ korea.ac.kr (M.C.); [email protected] (H.H.). † Korea University. ‡ Korea Basic Science Institute.

Figure 1. Structures of four peptides AD, AT, AAD, and AAT.

azido group: (1) the asymmetric azido stretching vibration has a large dipole strength, (2) the azido stretch frequency can be blue-shifted by about 14 cm-1 upon hydration, (3) its vibrational signature is in the transparent window (2000-2500 cm-1) free of protein IR absorptions, and (4) its size is comparatively small enough to minimally perturb the native structure of a protein.9-12 However, it has not been clarified yet whether the azido group can induce any conformational change via stereoelectronic effect when introduced into the β-carbon of alanine. The backbone conformations of short alanine-based peptides have been thoroughly studied by using various spectroscopic and theoretical calculation methods.13-72 It was suggested that alanine dipeptide (AD, Ac-Ala-NHMe, Figure 1), which is acetylated and amidated alanine containing two peptide bonds, adopts mainly polyproline II (PII) and some β-strand conformations in water.13-30 Even alanine tripeptide (AT, Ac-Ala-Ala-

10.1021/jp107359m  2010 American Chemical Society Published on Web 09/17/2010

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NH2, Figure 1) and slightly longer peptides exhibit nearly the same backbone conformation distribution.39-72 In contrast, the most stable backbone conformation of dipeptide in the gas phase has been found to be C7, where dipeptide has a seven-membered cyclic structure with an intramolecular hydrogen-bonding interaction between its two peptide bonds.31-38,73-75 Even in polar aprotic solvents such as chloroform and acetonitrile, some dipeptides were found to adopt the C7 structure because such intramolecular hydrogen-bonding interaction is not broken by solvent molecules. In water, however, the intramolecular hydrogen-bonding interaction between two peptide bonds is completely broken due to their stronger intermolecular hydrogenbonding interaction with solvent molecules. In addition, the intramolecular electrostatic interaction between two peptide bonds is diminished by the dielectric screening effect of water, leaving the intramolecular steric (repulsive) interaction dominant.28 This may be the physical basis for PII conformational preference of AD in aqueous solution. Here, we report the spectroscopic and computational studies on the conformation of β-azidoalanine (Aza) dipeptide (AAD, Ac-Aza-NHMe) and tripeptide (AAT, Ac-Aza-Aza-NH2) (Figure 1). To determine the backbone and side-chain conformations of a given peptide in solution, we employed NMR, amide I IR absorption, and electronic circular dichroism (CD) spectroscopies in combination with quantum chemistry calculations and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations. By comparison of experimental and theoretical results, the backbone conformation of AAD is found to deviate from the well-known PII structure but is rather close to the C7 structure in aqueous solution. We will show that the β-azido group can in fact play a role of directing the peptide backbone conformation toward the C7 structure despite the notably weakened intramolecular hydrogenbonding interaction between two peptide bonds upon hydration. As will be shown here, this is largely due to the azido gauche effect,1 which allows intramolecular electrostatic interactions between the β-azido group and two neighboring peptide bonds to be formed more easily. Thus, the β-azido group appears to be a potentially effective C7-conformation-directing element, which can be further implemented into other amino acids and polypeptides for their structural control. II. Experimental Section A. Materials. Peptides were prepared by methods reported previously (see the Supporting Information).8 All peptides were acetylated at the N terminus and amidated at the C terminus (see Figure 1). Note that two dipeptides AD and AAD contain the methylamide end, whereas two tripeptides AT and AAT contain the amide end at the C terminus. B. NMR Spectroscopy. All NMR experiments were carried out on a Varian VnmrS600 NMR spectrometer equipped with a 5 mm 1H{13C/15N} salt tolerant triple resonance cold probe. The WET and NOESY pulse sequences were employed for water suppression in one-dimensional (1D) and two-dimensional (2D) spectroscopies, respectively. Temperature was controlled with an L99 temperature controller (Varian) and a TC-84 nitrogen air cooler (FTS Systems). Chemical shifts were referenced to an internal standard: DSS (2,2-dimethyl-2silapentane-5-sulfonate, sodium salt) in D2O/H2O (1:9, pH 2: pH was adjusted by directly adding 0.3 N HCl), TMS (tetramethylsilane) in CD3CN, or solvent (1H: DMSO, δ 2.50 ppm; CHCl3, δ 7.27 ppm). 1D 1H NMR spectra were recorded on 10-15 mM AD and AAD in D2O/H2O (1:9, pH 2) at five temperatures (10, 20, 25,

Oh et al. 30, and 40 °C) and in DMSO-d6, CD3CN, and CDCl3 at 25 °C. A set of 25 000 (19 698) complex data points was collected, and 64 scans were averaged for aqueous (organic) solution. The original free induction decay was zero-filled to 262 144 points prior to Fourier transformation. The NMR data were analyzed with ACD/SpecManager (ACD/Labs). The 3J(HN,HR), 3J(HR,Hβ), and 2J(Hβ,Hβ) coupling constants were measured in 1D 1H spectra. The J values with error margins were obtained from peak fitting with a Gaussian+Lorentzian function. 2D 1H,1H NOESY spectra were recorded on 33.5 mM AD and 23.4 mM AAD in D2O/H2O (1:9, pH 2) at 25 °C. The pulse sequence was the standard NOESY in the Varian sequence library. The mixing time was optimized as 250 ms to minimize spin diffusion effects. A set of 4096 complex data points was collected in the t2 domain, and 128 t1 time increments were acquired. The spectra were recorded with 64 scans averaged and spectral widths were 9610.7 Hz in both dimensions. The NOESY data were analyzed with VnmrJ 3.0 software (Varian) and ACD/SpecManager (ACD/Labs). C. IR Spectroscopy. IR spectra were measured on a Bruker VERTEX 70 spectrometer equipped with a HgCdTe detector. To substitute the labile hydrogen atoms by deuterium atoms, the peptide/D2O solution was lyophilized twice on a SpeedVac (TermoSavant), and subsequently, it was dissolved in appropriate solvents. AD (AAD) was dissolved in D2O at 20.3 (15.0) mM and in DMSO at 12.4 (17.6) mM. AT (AAT) was dissolved in D2O at 11.8 (11.6) mM and in DMSO at 10.1 (12.9) mM. IR spectra were measured with a frequency resolution of 1 cm-1 in 100 scans using a CaF2 cell confined with a Teflon spacer (100 µm thickness). The cell chamber was purged with dry air to remove water vapor and CO2 inside the chamber. Temperature was controlled with an ATC-024 temperature controller (Harrick). All IR spectra were measured at 20 °C. D. CD Spectroscopy. CD spectra were measured on a JASCO J-815 spectrometer equipped with a 450 W Xe arc lamp. AD (AAD) was dissolved in H2O at 1.0 (1.0) mM and in CH3CN at 1.2 (1.6) mM. CD spectra were measured with a scan speed of 100 nm/min using a 110-QS quartz cell (Hellma) with a path length of 1 mm and averaged over 10 scans. Temperature was controlled with a PTC-423S/15 Peltier system (JASCO). CD spectra were measured at four temperatures (10, 20, 40, and 70 °C). CD spectra of DMSO and CHCl3 solutions were not examined because the solvent itself shows a strong UV absorbance in the information content region for peptide backbone structures.76 III. Quantum Chemistry Calculations and MD Simulations A. DFT Calculations. To study the relative energies of various AAD conformers, we carried out quantum chemistry calculations with density functional theory (DFT) at the B3LYP/ 6-31G* level in the Gaussian 03 suite.77-81 In fact, it is not easy to obtain the entire multidimensional potential energy surface of AAD molecules with respect to the φ and ψ backbone dihedral angles. Thus, we considered six representative backbone conformers with constant φ and ψ angles: seven-membered cyclic γ-turn (C7), right-handed R-helix (RR), π-helix (πH), antiparallel β-sheet (APB), parallel β-sheet (PB), and PII.13,32,82 For each of six backbone conformers, we also considered three side-chain conformers with the fixed χ1 side-chain dihedral angle: trans (t, χ1 ) 60°), gauche+ (g+, χ1 ) -60°), and gauche- (g-, χ1 ) 180°) (Figure 2).82 Thus, we considered 18 different conformers in total, but except for the three dihedral angles φ, ψ, and χ1, all the other internal degrees of freedom

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Figure 2. Newman projection of three staggered conformers viewed down the Cβ-CR bond axis in AAD. Note that trans (t), gauche+ (g+), and gauche- (g-) designations are given by reference to atoms C′-CR-Cβ-Nγ, whereas the χ1 side-chain dihedral angles are measured by reference to atoms HR-CR-Cβ-Nγ. In the IUPAC-IUB convention, χ1 is recommended to refer to the principal side-chain dihedral angle of atoms N-CR-Cβ-Nγ and to be positive when the bond to the front atom N (Nγ) requires rotation about the central bond in a clockwise sense for its eclipse to the bond to the rear atom Nγ (N).82 To conform to the IUPAC-IUB convention, t (χ1 ) 60°), g+ (χ1 ) -60°), and g(χ1 ) 180°) presented herein need to be changed to g- (χ1 ) -60°), t (χ1 ) 180°), and g+ (χ1 ) +60°), respectively. The 3J(HR,Hβ) values in Hz for each conformer are taken from Table 1 in ref 101 and used in eqs 1 and 2 in section IV.A.

TABLE 1: Relative Electronic Energies of AD and AAD Conformers in the Gas Phasea ∆EAADb (kJ/mol) conformers

t (χ1 ) 60°)

g+ (χ1 ) -60°)

g(χ1 ) 180°)

∆EADc (kJ/mol)

C7 RR πH APB PB PII

11.21 25.94 43.81 18.96 20.87 26.94

9.54 42.52 57.45 9.44 14.45 18.49

0 29.96 41.74 23.44 33.29 14.45

0 21.19 36.35 3.76 6.61 7.19

a Quantum chemistry calculation results on relative energies of representative conformers of AD and AAD (see Figure 3). See the detailed discussion in section III.A. b Relative energy of each conformer of an isolated AAD with respect to that of its C7/gconformer. The lowest energy conformer of AAD is C7/g- so that its energy is assumed to be zero. c Relative energy of each conformer of an isolated AD with respect to that of its C7 conformer.

were relaxed during the geometry optimization and energy calculation processes. The relative calculated electronic energies of 18 AAD conformers are summarized in Table 1. Here, the energy of the C7/g- conformer is assumed to be zero, since it is the lowest energy structure among them. In C7, πH, and PII, the g- conformer is the lowest energy structure, whereas the t conformer is the most stable in RR. In APB and PB, the g+ conformer is preferred. These results can be easily understood by examining the intramolecular electrostatic interaction patterns. In Figure 3, the geometry-optimized structures of 18 AAD conformers are depicted and the intramolecular electrostatic (or hydrogenbonding) interactions are highlighted with colored dashed lines. First of all, the C7/g- conformer, which is the lowest energy structure among all 18 conformers, has three strong intramolecular electrostatic interactions. There is an intramolecular hydrogen-bonding interaction between the two peptide bonds of AAD, which is the original element stabilizing the C7 structure even in AD (see the red dashed line in C7/g- of Figure 3). Additionally, the β-azido group forms two intramolecular electrostatic interactions with the N-terminal amide hydrogen atom and the C-terminal carbonyl oxygen atom: (1) H-bonding interaction between the N-terminal amide H atom and the N atom covalently bonded directly to ß-carbon (blue

dashed line) and (2) n f π* interaction between the carbonyl oxygen atom’s nonbonding orbital (n) and the azido group’s antibonding orbital (π*) (green dashed line). The azido gauche effect seems to allow the intramolecular electrostatic interactions between the β-azido group and two neighboring peptide bonds to be formed more easily. The number of such interactions also determines the relative stabilization energies of the other conformers. From these calculations, it is concluded that, when the solute-solvent interactions are excluded, the C7/g- conformer is close to the global energy minimum structure of AAD. This is largely due to the intramolecular cyclic hydrogenbonding interactions and the intramolecular electrostatic interactions originating from the azido gauche effect. B. QM/MM MD Simulations. Quantum chemistry calculations for various conformers of an isolated AAD provided useful information about the relative energies and detailed intramolecular interactions. However, to study possible solution structures of AAD in water, it is necessary to take into account the solvation effects on the peptide backbone structure. Therefore, we carried out quantum mechanical/molecular mechanical (QM/ MM) molecular dynamics (MD) simulations. Here, the solute AAD molecule was treated with semiempirical QM approximations, i.e., AM1 and PM383-85 and solvent TIP3P water molecules were treated classically. The detailed computational procedure can be found in ref 9. From the 1 ns 298 K QM/MM MD trajectories, the distributions of two dihedral angles φ and ψ are calculated and plotted in Figure 4. A notable feature is that, in both AM1 and PM3/MM MD simulations, the ψ angle distribution peaks around 0° and thus the backbone conformation of AAD in water is neither PII nor RR. Recently, we performed the same level of QM/MM MD simulations of AD in water and found that the backbone conformation of AD in water is a mixture of PII and RR (see Figure 4 in ref 22). Thus, these simulations suggest that the backbone conformation of AAD in water is strongly affected by the β-azido group. In addition to the φ and ψ angle distributions, we also examined the χ1 angle distribution (Figure 5). In the AM1/MM MD simulation, both g- and t conformers are greatly sampled, but the g+ conformer is not. In the PM3/MM MD simulation, the g- conformer is the dominant structure. Again, even in the aqueous solution structure of AAD, the intramolecular electrostatic interactions between the β-azido group and nearby two peptide bonds are important. In this section, we presented both quantum chemistry calculation and QM/MM MD simulation results of AAD. We found that the β-azido group can form strong intramolecular electrostatic interactions, which can affect the peptide backbone structure significantly. Next, we will present various experimental results, which show that the spectroscopic observations are also consistent with the above calculation results and that the azido gauche effect is manifest in AAD. IV. Experimental Results and Discussion A. NMR Spectra: O and χ1 Dihedral Angles Calculated from J Measurements. The distributions of the φ, ψ, and χ1 dihedral angles in peptides can be probed by NMR via spin-spin coupling constants. The coupling constants can be related to specific dihedral angles by Karplus relationships.86 The values of the measured coupling constants can provide direct information about the ensemble-averaged dihedral angles.23-26,61-70,87-95 The 3J(HN,HR) coupling constant for HN-N-CR-HR can be used to estimate the backbone dihedral angle φ (C′i-1-NiCRi-C′i).82 The 1H NMR spectra of AD (AAD) show a doublet

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Figure 3. Geometry-optimized structures of 18 AAD conformers in the gas phase. The dashed lines highlight the intramolecular electrostatic (or hydrogen-bonding) interactions between two peptide bonds (C′i-1 ) O · · · H-Ni+1Me or NisH · · · OdC′i, red), between the β-azido group (inner Nγ atom covalently bonded directly to β-carbon) and the N/C-terminal amide hydrogen atom (NtNsNγ · · · HsNi or NtNsNγ · · · HsNi+1Me, blue), or between the β-azido group (central N atom covalently bonded to inner γ-nitrogen) and the C-terminal carbonyl oxygen atom (NγsN · · · OdC′i, green). The calculated hydrogen bond lengths r in Å are the distances from the H atom to the atom H-bonded to the hydrogen. See the detailed discussion in section III.A. For quantitative information about the relative electronic energies of 18 AAD conformers, see Table 1. Color coding: white, hydrogen; gray, carbon; blue, nitrogen; red, oxygen.

Figure 4. Ramachandran plots, i.e., the distributions of the φ and ψ backbone dihedral angles for AAD in water. The plots were obtained from the 1 ns 298 K QM/MM MD trajectories.9 The solute AAD was treated semiempirical quantum mechanically, and the solvent water was treated classically with the TIP3P model. See the detailed discussion in section III.B.

for the acetyl-end amide proton, which has the 3J(HN,HR) values of 6.07 (7.38), 7.63 (8.36), 7.35 (8.06), and 7.88 (7.47) Hz at 25 °C in D2O/H2O (1:9, pH 2), DMSO-d6, CD3CN, and CDCl3, respectively (Table 2 and Figure S1 of the Supporting Information). The coupling constant can be related to the φ dihedral angle by a Karplus relationship, 3J(HN,HR) ) 9.44 cos2(φ - 60°) - 1.53 cos(φ - 60°) - 0.07.13,96,97 From the 3J(HN,HR) values for AD (AAD) at 25 °C in D2O/H2O (1:9, pH 2), DMSO-d6, CD3CN, and CDCl3, the corresponding φ angles are calculated to be -77 (-84), -86 (-90), -84 (-88), and -87 (-85) degrees, respectively (see Figure S1 of the Supporting Information). These results indicate that the backbone conformations of AD and AAD are close to either PII or C7. Note that the φ angle for AD in water is not significantly different from that in CDCl3 even though AD prefers PII in water but C7 in CDCl3. At present, it is not possible to determine whether the backbone

Figure 5. Distributions of the χ1 side-chain dihedral angles for AAD in water. The plots were obtained from the 1 ns 298 K QM/MM MD trajectories.9 In both AM1 and PM3/MM MD simulations, the most populated χ1 angle is 180°, which corresponds to the g- conformer. See the detailed discussion in section III.B.

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TABLE 2: Experimentally Measured NMR Coupling Constants of AD and AAD in Various Solvents at 25 °Ca solvents compounds AAD

coupling constants (Hz) 3

R b

J(H ,H ) J(HA,HB)c 3 J(HR,HA)c,d 3 J(HR,HB)c,d 3 J(HN,HR)b 2

AD

N

D2O/H2O (1:9, pH 2)

DMSO-d6

CD3CN

CDCl3

7.38 ( 0.001 12.95 ( 0.001 6.24 ( 0.001 4.66 ( 0.001 6.07 ( 0.001

8.36 ( 0.001 12.46 ( 0.001 5.34 ( 0.001 7.17 ( 0.001 7.63 ( 0.001

8.06 ( 0.011 12.62 ( 0.001 5.87 ( 0.001 4.99 ( 0.001 7.35 ( 0.002

7.47 ( 0.001 12.39 ( 0.001 5.39 ( 0.001 7.26 ( 0.001 7.88 ( 0.001

a

See experimental details in section II.B and the detailed discussion in section IV.A. Note that, in AAD, two β-protons, HA and HB, give two separate signals at higher (downfield) and lower (upfield) chemical shifts, respectively (see Figure 6). b See Figure S1 of the Supporting Information. c See Figure S2 of the Supporting Information. d See Table S1 of the Supporting Information.

Figure 6. Selected β-proton region of the 600 MHz 1H NMR spectrum of AAD in D2O/H2O (1:9, pH 2) at 25 °C. See experimental details in section II.B. Note that two β-protons giving two separate signals at higher (downfield) and lower (upfield) chemical shifts (δ) are indicated by HA and HB, respectively, each of which cannot be identified unequivocally to be a particular β-proton, Hβ1 or Hβ2, in Figure 2. See the detailed discussion in section IV.A.

conformations of AD and AAD in water are likely to be PII or C7 solely on the basis of the φ angle calculated from 3J(HN,HR). This suggests that some other complementary experimental evidence is needed for the ultimate structure determination. From the AM1 and PM3/MM MD trajectories in section III.B, we already presented the φ angle distributions for AAD in water (see Figure 4). These results can be used to calculate the 3 J(HN,HR) values, which are 7.87 and 7.12 Hz for the AM1 and PM3/MM MD simulations, respectively. However, if only parts of trajectories with χ1 ≈ 180° (g-) are considered, the resultant 3 J(HN,HR) values are 7.37 and 6.41 Hz. Note that the calculated 3 J(HN,HR) value of 7.37 Hz for AAD in water at 298 K is in close agreement with the experimentally measured value of 7.38 Hz for AAD in D2O/H2O (1:9, pH 2) at 25 °C. In the 1H NMR spectra of peptides, the 3J(HR,Hβ) coupling constant for HR-CR-Cβ-Hβ can provide information about the side-chain dihedral angle χ1 (HR-CR-Cβ-Nγ).82,90,91 The 1H NMR spectrum of AAD in D2O/H2O (1:9, pH 2) at 25 °C shows eight peaks around δ 3.70 ppm, which correspond to an AB quartet (Figure 6). In AAD, two β-protons, HA and HB, are chemically nonequivalent and thus exhibit different resonance signals around δ 3.74 and 3.65 ppm, respectively. Each of these two separate signals is further split into four peaks because the coupling constants between any two of the protons are different: 2 J(HA,HB) ) 12.95 Hz, 3J(HR,HA) ) 6.24 Hz, and 3J(HR,HB) ) 4.66 Hz (see Table 2). For example, the signal for HA is split into two by HB and again into two by HR. The two measured coupling constants 3J(HR,HA) and 3J(HR,HB) allow the relative populations of the three side-chain conformers g- (χ1 ) 180°), g+ (χ1 ) -60°), and t (χ1 ) 60°) to be determined. For the AAD side chain, the observed coupling constant can be expressed in terms of three conformer populations Pg-, Pg+, and Pt:

3.51Pg- + 4.14Pg+ + 12.32Pt ) 3J(HR, HX)

(1)

4.14Pg- + 12.32Pg+ + 3.51Pt ) 3J(HR, HY)

(2)

where Pg- + Pg+ + Pt ) 1 (see Figure 2).98-101 From these equations using the 3J(HR,Hβ) values for HX ) HA and HY ) HB (HX ) HB and HY ) HA), the conformer populations are calculated to be 60.9 (62.3), 8.7 (26.5), and 30.4 (11.2)% at 25 °C for Pg-, Pg+, and Pt, respectively, whereby a chemical shift for each of two β-protons, HA and HB, cannot be assigned unequivocally to a particular β-proton, Hβ2 or Hβ3, in AAD (see Figures 2 and 6). The order of these conformer populations in parentheses is consistent with that of energies obtained by ab initio calculations for the three side-chain conformers having all the C7 backbone structure in AAD (see section III.A and Table 1). On the other hand, the order of the other populations is consistent with that of side-chain conformer distributions obtained from QM/MM MD simulations of AAD in water (see section III.B and Figure 5). In both cases, however, the gconformer is found to be the predominant side-chain structure of AAD, which is consistent with both quantum chemistry calculation and QM/MM MD simulation results. The 3J(HR,Hβ) coupling constants of AAD in other solvents were also measured (see Table 2), and thereupon, the relative populations of the three side-chain conformers can be calculated (Table S1 of the Supporting Information). The 3J(HN,HR) coupling constants of AD and AAD in D2O/ H2O (1:9, pH 2) were measured for varying temperature (Figure 7). The 3J(HN,HR) values for AD (AAD) linearly increase with temperature: 5.89 (7.29) Hz at 10 °C and 6.27 (7.49) Hz at 40 °C. However, 3J(HN,HR) does not change much with temperature: 0.38 (0.20) Hz with a slope of 0.0132 (0.0066) Hz/°C. This result indicates that AD and AAD in water undergo negligible thermal transition between backbone conformations at least over this temperature range. Note that the thermal increase in 3J(HN,HR) for AAD is smaller than that for AD, suggestive of the higher structural rigidity of AAD in water. The 3J(HR,Hβ) coupling constants of AAD in D2O/H2O (1:9, pH 2) were also measured for varying temperature (Figure S2a of the Supporting Information). The 3J(HR,Hβ) values increase with temperature, although its increase with T is small. Thus, a thermal transition does not take place significantly between sidechain conformations of AAD in water. The temperaturedependent populations of the three side-chain conformers in water are plotted in Figure S2b-d of the Supporting Information. Backbone Conformation ObserWed from NOESY Spectra. As mentioned above, the backbone conformations of AD and AAD in water are suggested to be close to either PII or C7 by the φ angle calculated from 3J(HN,HR). To determine whether AD and AAD in water adopt a PII or C7 backbone conformation,

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Figure 7. 3J(HN,HR) coupling constants of AD (red circles, left y-axis) and AAD (blue circles, right y-axis) in D2O/H2O (1:9, pH 2) for varying temperature. See experimental details in section II.B.

we carried out 1H,1H NOESY experiments on AD and AAD in D2O/H2O (1:9, pH 2) at 25 °C (Figure 8). Figure 8a shows the structural difference between the PII and C7 backbone conformers of AAD. Note in particular that the distance between Hβ and HNMe of PII is shorter than that of C7 (about 3 vs 4 Å, see green double-headed arrows in Figure 8a). In the geometryoptimized structure of the C7/g- conformer obtained from ab initio calculations (see also section III.A and Figure 3), the distance between HNMe and HMeN of AD (AAD) is found to be 2.66 (2.33) Å. Hence, this distance is not much different depending on the backbone conformation and thus can be used as a fixed reference. Accordingly, the ratio of NOE intensities between Hβ and HNMe to that between HNMe and HMeN would be sensitive enough to distinguish between PII and C7 conformers (see NOE cross-peaks 4 and 6 in Figure 8b and c and Figures S3 and S4 of the Supporting Information).63 In the case of AD, we observed an NOE ratio of 0.720 for 250 ms mixing time, which corresponds to the 2.81 Å NOE distance between Hβ and HNMe (see NOE cross-peak 4 in Figure 8b and Figure S3 of the Supporting Information and Table 3). However, we detected little NOE signal for AAD under the same condition as AD (see lack of NOE cross-peak 4 in Figure 8c and Figure S4 of the Supporting Information and Table 3). These results indicate that the aqueous backbone conformation of AD is close to PII, whereas that of AAD clearly deviates from PII but is rather close to C7. The experimental NOE ratios and distances in AD and AAD for 450 ms mixing time were also measured (Figures S5 and S6 of the Supporting Information). B. IR Spectra: Backbone Conformation. Other than NMR, various vibrational spectroscopic methods such as IR absorption, Raman scattering, vibrational circular dichroism, and Raman optical activity have also been used to determine the secondary structures of polypeptides in solutions.52-60,102-110 Particularly, the amide I vibration, which is mainly the amide CdO stretch with a frequency range of 1600-1700 cm-1, has been considered to be one of the best marker modes. This is because its IR spectrum is quite sensitive to the peptide backbone structure and its vibrational transition dipole moment and Raman cross section are fairly large in comparison to those of other peptide vibrations.13-22,39-50 The amide I IR band itself cannot provide direct information about the dihedral angles such as φ, ψ, and χ1. However, its peak frequency and line shape are useful observables that can be used to distinguish at least two different solution structures, when only the side chain of a given amino acid is chemically modified, i.e., AD vs AAD. Herein, we therefore used the amide I IR spectroscopy to extract information about the solution structure of AAD and AAT. In Figure 9, the amide I IR spectra of four peptides in D2O and DMSO are plotted. First of all, the amide I IR band of AD

Oh et al. in D2O peaks at 1635 cm-1, whereas that of AAD in D2O peaks at 1643 cm-1 (Figure 9a). Thus, the amide I IR band of AAD is significantly blue-shifted by about 8 cm-1 in comparison to that of AD in D2O. On the other hand, the amide I IR peak frequency of AAD is relatively little blue-shifted by about 3 cm-1 () 1668-1665 cm-1) in comparison to that of AD in DMSO (Figure 9b). The blue shift difference between D2O and DMSO indicates that the amide I local mode frequencies, coupling constant, and local solvation environment are affected more greatly in water than DMSO by the β-azido group. This trend is even more clearly visible when the amide I IR spectra of AT are compared with those of AAT. The IR spectrum of AT is notably different from that of AAT in D2O (Figure 9c). More specifically, the amide I IR band of AT in D2O is a broad singlet, whereas that of AAT in D2O appears as a doublet in the high-frequency side. However, the amide I IR band of AT is similar to that of AAT in DMSO even though the relative intensities of individual (underlying) peaks change slightly (Figure 9d). Thus, it can be concluded that the solution structure difference between AD and AAD and between AT and AAT is greater in water than polar aprotic solvents like DMSO. This conclusion is partly validated by comparing the 3J(HN,HR) values for AD and AAD (see Table 2). The 3J(HN,HR) value for AD differs from that for AAD in water. However, the 3J(HN,HR) values for AD are quite close to those for AAD in CD3CN and CDCl3. The multiple fitting values for the amide I IR band are presented in Table S2 of the Supporting Information.50 Overall, we have a few notable observations and facts: (1) the backbone conformation of AD in water has been known to be predominantly PII, (2) the backbone conformation of AAD in water is found to be either PII or C7 from the NMR studies in section IV.A, and (3) as shown above, the amide I IR spectra of AAD and AAT are significantly different from those of AD and AT in water, respectively. From these experimental findings and theoretical calculation results, we suggest that AD and AT undergo conformational transition from PII to C7 in water upon β-azido-derivatization. Probably, further investigations of AAT with linear and two-dimensional IR spectroscopic techniques to measure the coupling constant between the two azido groups and to study its relationship with peptide backbone conformation will be of interest in the future. C. CD Spectra. CD spectra provide information about the backbone conformation of peptides. In Figure 10, the CD spectra of AD and AAD in H2O and CH3CN are plotted. The CD spectrum of AD in H2O shows a strong negative band at 195 nm and at 10 °C also has a weak positive band at 213 nm (Figure 10a). This is a characteristic line shape of alanine-based short oligopeptide having mainly PII and some β-strand conformations.61-66,111-116 With increasing temperature, the absolute CD intensity of the negative band at 195 nm decreases, but that of the positive band at 213 nm increases. This indicates that the relative population of β-strand increases with temperature even though PII is still dominant. Note that there is an isodichroic point at 205 nm, which is strong evidence that two distinguished conformers exist. The temperature-dependent CD spectrum of AAD in H2O shows a strong positive band at 185 nm, a weak negative band at 200 nm, a weak negative band at 216 nm, and an isodichroic point at 208 nm (Figure 10b). Interestingly, there is an additional very weak negative band at 275 nm at high AAD concentration (see the inset in Figure 10b). With increasing temperature, the absolute CD intensity of the negative band at 200 nm decreases, but that of the negative band at 216 nm increases. Note that the temperature dependence of such CD intensities in AAD is less significant than that in AD, suggestive of the higher backbone

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Figure 8. Geometry-optimized structures of the PII/g- and C7/g- conformers of AAD in the gas phase (a) and selected area of 1H,1H NOESY spectra of AD (b) and AAD (c) in D2O/H2O (1:9, pH 2) at 25 °C for 250 ms mixing time. For the geometry-optimized structures of the two AAD conformers, which are the same as those in Figure 3, see the detailed discussion in section III.A. The distance between Hβ and HNMe is indicated by a green double-headed arrow. For NOSEY spectra, which are also shown in Figures S3 and S4 of the Supporting Information, see experimental details in section II.B and the detailed discussion in section IV.A. For quantitative information obtained from NOE cross-peaks, see Table 3. NOE cross-peak assignment: 1, HN,HR; 2, HR,HNMe; 3, HN,Hβ; 4, Hβ,HNMe; 5, HAc,HN; 6, HNMe,HMeN.

TABLE 3: Experimental NOE Ratios and NOE Distances for AD and AAD in D2O/H2O (1:9, pH 2) at 25 °Ca NOE ratio NOE cross-peaks 1 2 3 4 5 6

N

R

H ,H HR,HNMe HN,Hβ Hβ,HNMe HAc,HN HNMe,HMeN

NOE distance (Å)

AD

AAD

AD

AAD

0.37 0.73 34.34 0.72 1.19 1

1.40 1.88 1.84 none 4.00 1

3.14 2.81 1.48 2.81 2.59 2.66b

2.20 2.10 2.11 none 1.85 2.33b

a Quantitative information obtained from Figure 8, where the mixing time for 2D 1H,1H NOESY spectra was 250 ms. See experimental details in section II.B and the detailed discussion in section IV.A. See also Figures S3, S4, and S7 of the Supporting Information. b A fixed reference obtained from the geometryoptimized structure of the C7/g- conformer, which was used to calculate NOE distances from their inverse one-sixth power relationship to NOE intensities.

conformational rigidity of AAD in water (see Figure 10a and b). This temperature-dependent behavior is similar to that discussed above with their coupling constants in section IV.A. From the UV-vis absorption spectrum of AAD (data not shown), we found that there are π-π* and n-π* transitions of the azido group at 185, 225, and 275 nm. Thus, the longest wavelength electronic transition appears as a very weak negative band at 275 nm in the CD spectrum. This band is solely

Figure 9. Amide I IR spectra of AD, AAD, AT, and AAT in D2O and DMSO at 20 °C. See experimental details in section II.C. For comparison, IR spectra are normalized. For quantitative information about the amide I IR bands, see Table S2 of the Supporting Information.

associated with the localized electronic transition of the azido group. The presence and temperature independence of this band suggest that the side-chain conformation of AAD is chiral and remains the same at 10-70 °C. The strong positive band at

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Oh et al. structure via its intramolecular electrostatic interactions with two neighboring peptide bonds, which are facilitated by the azido gauche effect. Thus, the β-azido group appears to be an effective C7-conformation-directing element, which may also be used to tune the structures of other amino acids and polypeptides. Acknowledgment. This work was supported by the NRF grant (No. 2009-0078897) and KBSI grant (T30401) to M.C., the KBSI grant (T30401) to G.-S.H., and the NRF grant (No. 2009-0075000) to H.H. We thank Dr. Jun-Ho Choi for providing us the QM/MM MD simulation trajectories.

Figure 10. CD spectra of AD and AAD in H2O and CH3CN for varying temperature. See experimental details in section II.D. The inset shows the CD spectra of 3 mM AAD in H2O, where the azido CD peak appears at 275 nm.

185 nm in the CD spectrum mainly originates from the electronic transition of the azido group. The temperature-dependent CD spectrum of AD in CH3CN shows a strong positive band at 185 nm, a strong negative band at 199 nm, and no notable isodichroic point at 200-225 nm (Figure 10c). With increasing temperature, mostly the absolute CD intensity of the negative band at 199 nm decreases. The CD spectral feature of AD in CH3CN, a less polar aprotic solvent, is a representative CD pattern of C7.13 Interestingly, the CD spectral line shape of AAD in H2O resembles that of AD in CH3CN at 200-250 nm. This suggests that the backbone conformation of AAD in water is close to C7 instead of PII. The temperature-dependent CD spectrum of AAD in CH3CN shows a weak negative band at 200-250 nm (Figure 10d). The CD spectral line shape at 200-250 nm does not change much as temperature increases, which suggests that the solution structure of AAD in CH3CN is homogeneous and C7. Intriguingly, the CD spectrum of AAD in CH3CN at 200-250 nm is notably similar to that of AAD in H2O at a high temperature. This suggests that the backbone conformation of AAD in water is a mixture of mainly C7 and some slightly distorted structure near the C7 basin. This finding is consistent with the AM1/MM MD simulation result even though the ensemble-averaged values of φ and ψ angles obtained from the MD trajectories are slightly different from those of an ideal C7 conformer. V. Summary In the present paper, we studied the backbone conformation of AAD and AAT in water and other polar aprotic solvents by using spectroscopic methods in combination with quantum chemistry calculations and QM/MM MD simulations. From the 1 H NMR coupling constants and 1H,1H NOESY experimental results, it was shown that AAD in water mainly adopts a C7 rather than PII backbone conformation and prefers the g- sidechain conformer. From the comparative investigations of amide I IR absorption and CD spectra, the backbone conformation of AAD in water is found to deviate from PII but is rather close to C7. Thus, the backbone conformation of AAD differs from that of AD, which is mainly PII in water. The underlying origin of the backbone conformational difference between AAD and AD in water was elucidated by quantum chemistry calculations. It was found that the β-azido group can actively participate in directing the peptide backbone conformation toward the C7

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