9768
J. Phys. Chem. A 2010, 114, 9768–9773
Unambiguous Assignment of Vibrational Spectra of Cyclosporins A and H† Zheng-wang Qu,*,‡ Hui Zhu,§ and Volkhard May‡ Humboldt UniVersita¨t zu Berlin, Institut fu¨r Physik, AG Photobiphysik, Newtonstrasse 15, D-12489 Berlin, Germany, and Freie UniVersita¨t Berlin, Institute for Mathematics, Arnimallee 6, D-14195 Berlin, Germany. ReceiVed: March 11, 2010; ReVised Manuscript ReceiVed: April 15, 2010
Vibrational infrared and circular dichroism spectra of two cyclosporins A and H are predicted by density functional theory calculations using the nuclear magnetic resonance proposed structure in chloroform. Spectral signatures in the important amide II, I, and A regions are identified for typical peptide secondary structures including type II′ β-turn, antiparallel β-sheet, inverse γ-turn, N-methylated peptide bond, and side-chain H-bond. Our theoretical spectra agree very well with available experimental data in nonpolar media and unambiguously clarify their controversial vibrational assignment. The new insights into the spectral signatures of secondary structures can be very useful for peptide conformation analysis in general. Introduction Cyclosporine A (CsA, C62H111N11O12) is an immunosuppressive drug1 widely used in organ transplants, which can bind to the intracellar receptor protein cyclophilin (CyP) and the antiCsA antibody fragment (Fab).2-4 A considerable amount of structural information is available concerning both free and bound forms of cyclosporins.2-15 Scheme 1 shows the NMRproposed6 free CsA structure in chloroform. It is a cyclic peptide containing 11 amino acid residues in the following sequence: MeBmt1, Abu2, Sar3, MeLeu4, Val5, MeLeu6, Ala7, D-Ala8, MeLeu9, MeLeu10, and MeVal11. Here the chiral R-carbons of all residues (except for D-Ala8 and nonchiral Sar3) adopt L-conformation and seven residues are N-methylated. Cyclosporin H (CsH) adopts the same cyclic residue sequence but with D-conformation for site 11 (D-MeVal11) instead. Even such a little change in chirality already reduces the immunosuppressive activity of CsH to near zero. An understanding of the bioactivity of cyclosporin requires knowledge of their steric structures under different conditions. In nonpolar solvents such as chloroform, NMR studies6-8 proposed that there is an intramolecular side-chain 1OH · · · OC1 H-bond within MeBmt1 of CsA, instead of an intermolecular H-bond from the MeBmt1 OH group to another CsA or structural water molecule found in crystal.5,8 Infrared (IR) spectra studies9,10 in polar solvents such as acetonitrile and DMSO suggested that several conformations of cyclosporins may exist in equilibrium with mostly retained backbone structures. Other conformations in solution have also been suggested, for example, with partial cis-trans isomerization of peptide bonds when interacting with metal ions such as Li+, Ca2+, and Mg2+,11-15 with an intramolecular 1OH · · · OC3 H-bond in the complex with dimethyl isosorbide,5 and with much more open structures3,4 when binding to the receptor protein CyP or the anti-CsA antibody fragment (Fab). The unusual MeBmt1 residue within CsA is known to be important for its bioactivity. There is extensive interest to understand the spectral signatures of peptide secondary structures like β-sheet, β-turn, and †
Part of the “Reinhard Schinke Festschrift”. * To whom correspondence should be physik.hu-berlin.de. ‡ Humboldt Universita¨t zu Berlin. § Freie Universita¨t Berlin.
addressed,
zheng@
SCHEME 1: Typical Secondary Structures within the NMR-Proposed Structure of Cyclosporin A (C62H111N11O12, 196 atoms) in Chloroforma
a The ring structure is labeled by the residue sites of the 11 amino acids: MeBmt1, Abu2, Sar3, MeLeu4, Val5, MeLeu6, Ala7, D-Ala8, MeLeu9, MeLeu10, MeVal11. Five polar hydrogen atoms are shown in red with possible intramolecular H-bonds shown as dashed lines. The backbone (φ, ψ, and ω) and side-chain (χ) dihedrals are displayed for MeLeu9 as example.
γ-turn.16,17 As midsize peptides, cyclosporins can adopt typical secondary structures with new features such as side-chain OH · · · OC H-bonds and N-methylated peptide bonds especially in nonpolar media. Thus, they represent interesting models for combined experimental and theoretical studies. The NMRproposed6 free CsA structure in chloroform offers a good starting point for theoretical simulations. Early studies9,10 of IR spectra have provided some qualitative information on the structures of free cyclosporins in different solvents. However, there is still a lack of clear spectral assignments especially for turn structures and new structural features like N-methylation and side-chain H-bonds. For example, a single strong IR peak either around 1620 cm-1 (refs 9, 12, and 17) or around 1660 cm-1 (ref 10) in chloroform has been tentatively assigned to the tight 8NH · · · OC6 γ-turn H-bond within CsA, while a weak vibrational IR and circular dichroism (VCD) peak around 1645 cm-1 has been suggested11 by recent density functional theory (DFT) calculations. To analyze the experimental IR and VCD spectra of various cyclosporins, backbone fragments from X-ray crystal structures are used while bulky side-chains and possible sidechain H-bonds are neglected.11 Different from the NMRproposed6 structure, no intramolecular 1OH · · · OC1 H-bond is
10.1021/jp102206z 2010 American Chemical Society Published on Web 04/28/2010
Spectral Assignment of Cyclosporins found in the crystal forms of CsA as well as CsH, and even the 7NH · · · OC11 and 8NH · · · OC6 H-bonds are broken in the CsH crystal structure.5,8,10 Since measured spectra in the amide II, I, and A regions are very sensitive to intramolecular H-bonds and N-methylation, reliable theoretical simulations are still required to identify the spectral signatures of typical secondary structures involved in free cyclosporins in solution. In this work, starting from the NMR-proposed6 CsA structure in nonpolar media, reliable DFT calculations are performed to explicitly identify the IR and VCD signatures of typical secondary structures involved in both CsA and CsH molecules. On the basis of more realistic molecular models taking both peptide backbone and bulky side chains into account, our results provide unambiguous vibrational assignment to experimental data and new insights into the spectral signatures of peptide secondary structures. 2. Computational Method All DFT calculations are performed using the Gaussian 03 program package18 with the accurate hybrid B3LYP19,20 functional. Each of the real CsA and CsH molecules contains 196 atoms, which is too expensive for reliable frequency analysis using a large basis set. The two-layer ONIOM21 model (B3LYP/ 6-31G(d): B3LYP/STO-3G) is adopted to reduce some computational efforts, using higher B3LYP/6-31G(d) calculation22,23 for a model system containing only the most important part but lower B3LYP/STO-3G calculation24 for the remaining part of the real molecule. In each case, the model system is chosen to include the peptide backbone and the close parts of alkyl side chains only up to the β-carbons, while the remaining part consists of the far parts of long side chains away from the β-carbons. In this way, the number of atoms to be treated at higher calculation is reduced from 196 into 129. In each case, the ONIOM energy of the real molecule (E(ONIOM,real)) is taken as the energy of the model system from higher calculation (E(high,model)) corrected by the energy of the remaining part from low calculation (E(low,real) - E(low,model))
E(ONIOM,real) ) E(high,model) + (E(low,real) - E(low,model)) With the ONIOM model, the electronic properties of the important peptide backbone and its close side chains are treated at high accuracy, while the steric interactions from far parts of bulky side chains are still reasonably included. Starting from the NMR-proposed6 structure, the possible geometries of the CsA molecule are fully optimized using the above ONIOM model. By inverting the R-carbon conformation at site 11 into D-conformation, the geometries of the CsH molecule are also optimized from similar initial structures. After geometry optimization, harmonic frequencies are calculated together with both the IR intensity and VCD rotational strength25 of each vibrational mode. For better comparison with the experimental IR and VCD spectra9,11 taken in nonpolar media, all frequencies are scaled with a common factor of 0.955. The predicted IR and VCD intensities are further convoluted with a Gaussian line shape of 15 cm-1 fwhm. Note that for each vibrational mode the IR intensity is positive, while the VCD strength, defined as the absorbance difference of left and right circularly polarized light, could be positive or negative. 3. Results and Discussion 3.1. Structural Features of CsA. The NMR-proposed6 CsA structure (Scheme 1), which is the starting point of our DFT calculations, shows some important structural features. The
J. Phys. Chem. A, Vol. 114, No. 36, 2010 9769 conformation of each residue can be defined by the backbone (φ, ψ, and ω) and side chain (χ) dihedrals: The dihedrals φ and ψ define flexible backbone conformations before and after an R-carbon, the ω gives the somewhat rigid peptide bond conformation (around 0° for cis and 180° for trans), and the χ characterizes flexible side-chain rotations. By definition, there is no dihedral χ for Sar3, Ala7, and D-Ala8. CsA contains five intramolecular H-bonds that define some typical peptide secondary structures: (i) the type II′ β-turn centered at Sar3 and MeLeu4 (with (φ, ψ) around (79°, -108°) and around (122°, 30°), respectively) and closed by downward 5NH · · · OC2 H-bond, (ii) the twisted antiparallel β-sheet extended by 2NH · · · OC5 and 7NH · · · OC11 H-bonds, (iii) the inverse γ-turn centered at Ala7 (with (φ, ψ) around (-67°, 54°)) and closed by 8NH · · · OC6 H-bond, and (iv) the turnlike structure within MeBmt1 closed by the side-chain 1OH · · · OC1 H-bond. The intermolecular 1OH · · · OC9 H-bond found in tetragonal CsA crystal8 is broken in solution. Seven peptide bonds are Nmethylated (NMe-CO), leading to the cis-9,10 peptide bond in the loop structure. As will be explained below, N-methylated peptide bonds show quite different spectral signatures from normal NH-CO peptide bonds, which must be taken into account when discussing peptide secondary structures. There are three natural pairs of close side-chain interactions due to the W-shaped β-sheet structure (see Figure 1 below): the top pair (MeBmt1, MeLeu6) and two bottom pairs (Abu2, Val5) and (Ala7, MeVal11). To avoid strong steric hindrance, the far side-chain parts of MeBmt1 and MeLeu6 stay in the left β-sheet groove near Val5 and the right groove near Ala7, respectively. These structural features are important when trying to understand the vibrational spectra of cyclosporins in nonpolar solutions. 3.2. DFT Optimized CsA and CsH Structures. Figure 1 shows the top and side view of the optimized geometries of CsA and CsH molecules. The most important backbone (φ and ψ) and side-chain (χ) dihedrals of each residue are listed in Table 1. The DFT-optimized CsA geometries with five tight intramolecular H-bonds closely resemble the NMR-proposed6-8 CsA structure in nonpolar media. It retains all the proposed6 secondary structures including the type II′ β-turn, β-sheet, tight inverse γ-turn, side-chain 1OH · · · OC1 H-bond, loop structure with cis-9,10 peptide bond, and the side-chain orientations of bulky MeBmt1 and MeLeu6 side-chains. The DFT-optimized CsA backbone dihedrals are very close (with standard deviation of 13°) to those from NMR-restrained MD simulations.6 The DFT-optimized χ dihedrals of residues 1, 6, 9, 10, and 11 are rather consistent with those from NMR-restrained MD simulations,6 though several side-chain orientations are possible in solution. With a D-conformation at site 11 in CsH molecule instead, the whole D-MeVal11 side chain stays more parallel (rather than perpendicular) to the β-sheet structure. Thus it is further away from the Ala7 side chain with fewer steric interactions. The new chirality at site 11 within CsH leads to a large change of 26° with respect to the φ dihedral at site 11 as well as small changes of 6°, 6°, and 4° of the ψ dihedrals at sites 11, 10, and 7, respectively, with other backbone dihedrals remaining almost the same as those found in the CsA molecule. 3.3. Vibrational Assignments. Figure 2 shows the vibrational assignment of DFT-predicted IR and VCD spectra related to the CsA and the CsH molecule in the respective amide II, I and A regions around 1500, 1650, and 3300 cm-1. Table 2 lists the detailed vibrational frequencies, IR and VCD intensities and assignments. Some vibrations of alkyl side-chains with rather low IR intensities are artificially blue-shifted into the low frequency side of the amide I region due to the applied low-
9770
J. Phys. Chem. A, Vol. 114, No. 36, 2010
Qu et al.
Figure 1. Top and side views of the optimized geometries of cyclosporins A and H. Some H-bond proton-acceptor distances are displayed (in Å). The C, N, and O atoms are shown by gray, blue, and red spheres. Five polar H-atoms within the OH and NH groups are shown by green spheres while nonpolar H-atoms are hidden for clarity.
TABLE 1: DFT-Optimized Backbone (O and ψ) and Side-Chain (χ) dihedrals (in deg) of the 11 Amino Acid Residues of CsA and CsH residues structure CsA CsH CsAa
a
φ ψ χ φ ψ χ φ ψ χ
1
2
3
4
5
6
7
8
9
10
11
-87 129 -59 -90 126 -62 -89 112 -77
-112 90 -178 -111 90 -178 -97 100 -70
72 -124
-113 24 -56 -115 25 -57 -122 30 -151
-90 127 174 -90 127 174 -104 123 -61
-109 104 179 -110 104 180 -82 88 -178
-85 59
77 -124
-86 55
79 -125
-67 54
80 -137
-121 100 -55 -120 99 -55 -125 116 -60
-145 72 -164 -143 66 -162 -131 86 -148
-100 126 -50 -74 120 -63 -120 133 -60
72 -124 79 -108
NMR-restrained MD simulations from ref 6.
level treatment. This has been mostly filtered out by introducing a combined intensity threshold of 50 km/mol for IR and 25 × 10-44esu2cm2 for VCD. The spectral assignments are labeled by the major residue sites involved in the corresponding vibrational modes. For qualitative discussion, the IR peaks with intensity larger than 100 km/mol (smaller than 50 km/mol) are assumed to be strong (weak). Similarly, the VCD peaks with strength larger than 100 × 10-44esu2cm2 (smaller than 50 × 10-44esu2cm2) are assumed to be strong (weak), using (+) for positive and (-) for negative VCD peaks. 3.3.1. Spectral Signatures in the Amide II Region. The amide II vibrations around 1500 cm-1 are mainly due to N-H in-plane bending with out-of-phase C-N stretching contributions within four backbone NH-CO peptide bonds, and thus labeled by the residue sites (Abu2, Val5, Ala7, and D-Ala8) of involved NH groups. The amide II bands can be blue-shifted (to higher frequency) by strong H-bonds from N-H groups of the peptide bonds. For the CsA molecule, the strong IR (strong (-) VCD) peaks at 1528 and 1519 cm-1 are due to Abu2 and Ala7 of the twisted β-sheet, respectively. The IR (strong (+) VCD) peak at 1496 cm-1 is originated by Val5 of the type II′ β-turn. The IR (weak (+) VCD) peak at 1511 cm-1 is caused by D-Ala8 of the inverse γ-turn. Much weaker IR and VCD peaks may also appear below 1490 cm-1 due to N-methyl deforming and/or C-N stretching
vibrations, which are less important for our conformation analysis. For the CsH molecule with D-MeVal11 instead, the spectral signatures due to Abu2 and Val5 remain unchanged. However, the amide II vibrations of Ala7 and D-Ala8 are strongly coupled, leading to the strong IR (strong (-) VCD) peaks at 1515 cm-1 due to D-Ala8 with in-phase Ala7 contribution and the strong IR (medium (+) VCD) peak at 1514 cm-1 due to Ala7 with an out-of-phase D-Ala8 contribution. 3.3.2. Spectral Signatures in the Amide I Region. The amide I vibrations around 1650 cm-1 are mainly due to CdO stretching with minor out-of-phase C-N stretching and CCN deformation (and minor NsH in-plane bending if present). Thus they are labeled by the sites of involved CdO groups. The amide I bands are strong and very sensitive to backbone conformations (they are frequently used for peptide secondary structure analysis). The amide I bands can be red-shifted by structural features like H-bonds to CdO and N-methylation that may stabilize the ionic resonance structure (with more CsO single-bond and CdN double-bond characters) of the peptide bond. For the CsA molecule, at the blue side of amide I region, the strong IR (strong (+) VCD) peak at 1676 cm-1 and the strong IR (strong (-) VCD) peak at 1678 cm-1 are due to free NH-CO peptide bonds of Ala7 within the γ-turn and MeLeu4 within the β-turn, respectively. In the central amide I region, the medium IR (weak (-) VCD) peak at 1637 cm-1 and the weak
Spectral Assignment of Cyclosporins
J. Phys. Chem. A, Vol. 114, No. 36, 2010 9771
Figure 2. Vibrational assignments of the predicted IR and VCD spectra of (a) cyclosporin A and (b) cyclosporin H. The computed spectra are presented by solid lines with the original intensities indicated by dots. The major residue involved in each vibrational mode is indicated by the corresponding residue sites at first, with the in-phase and out-of-phase contributions from minor residue indicated by + and -, respectively.
TABLE 2: Frequencies (in cm-1), IR Intensities (in km/mol), VCD Strengths (in 10-44 esu2 cm2), and Assignments of Important Vibrations of CsA and CsH in the amide II, I and A regions. The assignments are given by the major residues involved CsA
CsH freq
IR
VCD
assigna
NH Bending Modes β-turn H-bond γ-turn H-bond β-sheet H-bond β-sheet H-bond
1496 1514 1515 1531
151 273 122 172
245 85 -242 -120
5 8-7 7+8 2
11 2 5+1 8 1-5 6 9-10 3 10+9 7 4
CdO Stretching Modes β-sheet H-bond, NMe β-turn H-bond, NMe β-sheet H-bond, NMe free, NMe turn-like 1OH · · · OC1 γ-turn H-bond cis-peptide, NMe β-turn, first free, NMe free, NMe γ-turn, free β-turn, second free
1585 1619 1629 1638 1637 1645 1648 1648 1653 1673 1678
146 486 252 247 71 52 312 218 203 250 222
-58 -70 -172 117 62 10 -335 200 22 201 -83
11 2 5+1 8-9 1-5 6 9-10 3 10+9 7 4
2 8 7 5 1OH
N-H Stretching Modes β-sheet H-bond γ-turn H-bond β-sheet H-bond β-turn H-bond turn-like 1OH · · · OC1
3283 3332 3342 3374 3449
391 210 247 219 114
44 -216 261 -34 112
2 8+7 7-8 5 1OH
freq
IR
VCD
assign
1496 1511 1519 1528
166 281 122 172
204 5 -180 -112
5 8 7 2
1618 1619 1629 1636 1637 1645 1646 1649 1650 1676 1678
168 183 230 297 60 37 239 332 144 203 265
-245 -220 -184 116 -14 57 -259 189 109 175 -119
3279 3327 3354 3373 3439
428 189 223 222 117
43 -62 133 -47 112
a
secondary structure
b
a The assignments are given by the major residues involved. The major residue at first, with the in-phase and out-of-phase contributions from minor residue indicated by + and -, respectively. b For the major residue only. Some short notations used here: NMe, N-methylated; free, without H-bond to peptide carbonyl; β-sheet, antiparallel β-sheet; β-turn, type II′ β-turn; γ-turn, type inverse γ-turn.
IR (medium (+) VCD) peaks at 1645 cm-1 are mainly cause by the H-bonded NH-CO peptide bonds of MeBmt1 within 1OH · · · OC1 turn and MeLeu6 within the γ-turn, respectively. Note that the amide I vibrations of Val5 and MeBmt1 are somewhat coupled together. The strong IR (strong (+) VCD) peaks at 1636, 1649, and 1650 cm-1 and the strong IR (strong (-) VCD) peak at 1646 cm-1 are mainly originated by the N-methylated NMe-CO peptide bonds of D-Ala8, Sar3, MeLeu10, and MeLeu9, respectively, with the negative VCD feature likely related to the cis-9,10 peptide conformation. Note
that the amide I vibrations of MeLeu9 and MeLeu10 are strongly coupled together. Finally, at the red side of the amide I region, the strong IR (strong (-) VCD) peaks at 1618, 1619, and 1629 cm-1 are mainly due to the H-bonded and N-methylated NMeCO peptide bonds of MeVal11, Abu2, and Val5, respectively, within the β-sheet and β-turn structures. For the CsH molecule with D-MeVal11 instead, the strong IR (medium (-) VCD) peaks at 1585 and 1619 cm-1 are mainly due to D-MeVal11 and Abu2, respectively, with the former being evidently red shifted by 33 cm-1. The medium IR (medium (+)
9772
J. Phys. Chem. A, Vol. 114, No. 36, 2010
VCD) peak at 1637 cm-1 and the medium IR (weak (+) VCD) are due to MeBmt1 and MeLeu6, respectively, with changed VCD sign likely due to new D-MeVal11 side-chain orientation nearby. The amide I vibrations of MeLeu9 and MeLeu10 are still strongly coupled together, leading to a strong IR (strong (-) VCD) peak at 1648 cm-1 (mainly due to MeLeu9 with outof-phase MeLeu10 contribution) and a strong IR (weak (+) VCD) peak at 1653 cm-1 (due to MeLeu10 with in-phase MeLeu9 contribution). Other spectral signatures remain unchanged. 3.3.3. Spectral Signatures in the Amide A Region. The amide A vibrations around 3300 cm-1 are mainly due to N-H stretching; thus they are labeled by the residue sites (Abu2, Val5, Ala7, and D-Ala8) of the involved NH groups. The amide A bands can be red-shifted by strong H-bonds from the N-H groups. The stretching vibration of free or weakly H-bonded NH and strongly H-bonded MeBmt1 OH groups may be shown up at the blue side of the amide A region above 3400 cm-1. For the CsA molecule, the strong IR (weak (+) VCD) peak at 3279 cm-1 and the strong IR (strong (+) VCD) peak at 3354 cm-1 are due to the H-bonded NH groups of Abu2 and Ala7 within the β-sheet structure. The strong IR (medium (-) VCD) peak at 3327 cm-1 and the strong IR (weak (-) VCD) peak at 3373 cm-1 are due to the H-bonded NH groups of D-Ala8 within the γ-turn and Val5 within the β-turn, respectively. Finally, the strong IR (strong (+) VCD) peak at 3439 cm-1 is due to the 1OH · · · OC1 H-bond within MeBmt1. For the CsH molecule with D-MeVal11 instead, the amide A vibrations of Ala7 and D-Ala8 are strongly coupled, leading to the strong IR (strong (-) VCD) peak at 3332 cm-1 and the strong IR (strong (+) VCD) peak at 3342 cm-1. Other spectral signatures remain unchanged. 3.4. Comparison with Experiment. Well-resolved IR spectra9 of CsA and CsH molecules in the amide II and I regions have been obtained in nonpolar carbon tetrachloride (CCl4) and chloroform solvents using the Fourier self-deconvolution bandnarrowing method. In more polar solvents like acetonitrile and DMSO, the IR peaks become much broader with less vibrational structures, possibly due to more disrupted secondary structures.9,10 For this reason, the most resolved IR spectra9 in CCl4 are taken for comparison with our DFT-predicted spectra in Figure 2. Excellent agreement is found concerning both peak frequencies and more detailed line shapes, leading to unambiguous vibrational assignments for the experimental spectra. In the amide II region that is mainly due to N-H bending, three peaks are experimentally resolved for both CsA and CsH spectra: The peak around 1490 cm-1 is due to the type II′ β-turn H-bond (Val5), the middle peak around 1515 cm-1 is caused by the inverse γ-turn (D-Ala8) as well as the antiparallel β-sheet (Ala7) H-bonds, and the peak around 1530 cm-1 is determined by the antiparallel β-sheet (Abu2) H-bond. In the strong amide I region, mainly caused by the CdO stretching, four peaks are well resolved in the experimental CsA spectrum:9 two strong peaks at 1625 and 1638 cm-1 and two weaker peaks at 1676 and 1685 cm-1. By comparison with our DFT calculations, the first strong peak is actually due to three N-methylated peptide bonds involved in the type II′ β-turn H-bond (Abu2) and the antiparallel β-sheet H-bonds (MeVal11 and Val5). The second strong peak is mainly caused by the free N-methylated peptide bonds within the loop (D-Ala8, MeLeu9, MeLeu10) and type II′ β-turn (Sar3) structures, with minor contributions from the unmethylated peptide bonds involved in the inverse γ-turn (MeLeu6) and the turn-like 1OH · · · OC1 (MeBmt1) H-bonds. The last two weaker peaks are originated by two free unmethylated peptide bonds involved in the inverse
Qu et al. γ-turn (Ala7) and type II′ β-turn (MeLeu4) structures. Five peaks are well resolved in the experimental CsH spectrum.9 Compared with the CsA spectrum, the first peak at 1616 cm-1 is new while the second peak becomes weaker than the third peak. This evident change of the peak pattern for CsH spectrum is well reproduced by our DFT calculations: the IR peak due to the antiparallel β-sheet H-bonds (MeVal11) is evidently red-shifted into the new peak at low frequency and thus separated from the second peak with reduced intensity. The last three peaks have the same vibrational assignments as those observed in the CsA spectrum, though their relative intensity patterns are slightly different. In the amide I region, low-resolution VCD spectra for various cyclosporins are also available in chloroform.11 Only two VCD peaks in the CsA and CsH spectra are well-resolved:11 the first strong (-) peak around 1620 cm-1 and the second broad (+) peak around 1645 cm-1. The VCD intensity of the first (-) peak in the CsH spectrum is evidently lower than that in the CsA spectrum. These spectral features are well reproduced by our DFT calculations. According to the discussion in section 3.3.2, the first (-) VCD peak around 1620 cm-1 is due to three N-methylated peptide bonds involved in the β-turn (Abu2) and β-sheet (MeVal11 and Val5) H-bonds that consistently produce negative VCD features at low frequency. The second broad (+) peak around 1645 cm-1 follows from a more detailed balance between negative VCD features (MeLeu9, MeLeu4) and other positive VCD features. However, more detailed analysis is prevented by the low-resolution of experimental spectra. In the amide A region around 3300 cm-1 mainly caused by the N-H stretching, only low-resolution IR and VCD spectra are available from experiments in chloroform.11 The observed spectra in the amide A region are much weaker and broader than those in the amide I region,11 making quantitative assignment more difficult. Even then, according to the discussion in section 3.3.3, our DFT calculations clearly show that for the CsA and CsH molecules the broad IR ((+) VCD) peak around 3420 cm-1 is due to the turn-like intramolecular 1OH · · · OC1 H-bond within MeBmt1 while other spectral features below 3400 cm-1 follow from H-bonded peptide bonds. The strong IR (strong (-) VCD) peak around 3320 cm-1 and the IR shoulder ((+) VCD peak) around 3280 cm-1 observed for CsA are most likely due to the inverse γ-turn (D-Ala8) H-bond and the antiparallel β-sheet (Abu2) H-bond, respectively. For CsH molecule, the amide A vibrations of the inverse γ-turn (D-Ala8) and the β-sheet (Ala7) H-bonds are strongly coupled together, leading to very weak VCD peak around 3320 cm-1 due to intensity cancellation. No peaks above 3500 cm-1 are observed in our DFT calculations. The experimentally measured weak peaks above 3500 cm-1 are most likely due to impurities such as water molecules. As discussed above, an excellent agreement between our DFT calculations and experimental IR and VCD spectra9,11 could be achieved, especially in the strong amide I region, which results in an almost unambiguous vibrational assignment. Further vibrationally resolved VCD spectra (possibly from low temperature experiment in CCl4) would be very helpful for a further and more detailed assignment. The excellent agreement between theory and experiment strongly confirms that the CsA and CsH molecules mainly adopt the NMR-proposed6 structure with five intramolecularH-bonds(5NH · · · OC2,2NH · · · OC5,7NH · · · OC11, 8NH · · · OC6 and 1OH · · · OC1) in nonpolar solvents such as CCl4 and chloroform. In their crystal forms,5,8,10 some of these intramolecular H-bonds are broken (1OH · · · OC1 for CsA and CsH, and 7NH · · · OC11 and 8NH · · · OC6 for CsH), which will
Spectral Assignment of Cyclosporins lead to a quite different intensity pattern in spectra. This could be the main reason for the qualitative failures11 of the earlier DFT analysis (especially for CsH) based on backbone fragments of crystal structures. The predicted spectra cannot even qualitatively reproduce the observed9 intensity patterns and the strong IR ((+) VCD) peak around 3420 cm-1 in the amide A region. Experimentally, the assignment of peptide secondary structures is mostly based on some observations from earlier studies of proteins and model peptides.16,17 In the amide I region, absorption bands below 1645 cm-1 and above 1660 cm-1 are normally assigned to strongly H-bonded and to free peptide bonds, respectively. However, new structural features like N-methylation and side-chain 1OH · · · OC1 H-bond make the situation much more complicated. In particular, the IR peaks at 1625 and 1616 cm-1 in chloroform were tentatively assigned to the γ-turn H-bond of CsA and CsH.9,12 More recently, the IR peaks at 1661, 1675, and 1682 cm-1 for CsA in chloroform were related10 to the γ-turn H-bond, β-turn H-bond, and 1OH · · · OC1 H-bond, respectively. However, our DFT calculations clearly show that the low-frequency peaks around 1620 cm-1 are due to N-methylated peptide bonds involved in β-turn and β-sheet H-bonds while the high-frequency peaks around 1680 cm-1 are due to free unmethylated peptide bonds involved in β-turn and γ-turn structures. The IR and VCD features caused by the γ-turn and turn-like 1OH · · · OC1 H-bonds are very weak and should appear around 1640 cm-1. Moreover, there are some experimental attempts17 to relate single spectral signature for identifying secondary structures. In fact, multiple signatures are possible for all kinds of secondary structures. For example, a typical β-turn structure involving three peptide bonds shows three peaks in the amide I region, with each affected by more structural details like N-substitution and β-turn type. Thus, caution must be taken to reliably assign experimental spectral signatures to typical peptide secondary structures. Close interplay between experiment and theory can be very useful for this purpose. 4. Conclusions The spectra of cyclosporins A and H are predicted by reliable DFT calculations and compared with available experimental data. Excellent agreement between experiment and theory strongly confirms that both cyclosporins mainly adopt a structure very similar to the NMR-proposed structure with five intramolecular H-bonds. Unambiguous spectral signatures in the important amide II, I, and A regions are identified for typical peptide secondary structures including type II′ β-turn, antiparallel β-sheet, inverse γ-turn, N-methylated peptide bond, and side-chain H-bond. Some controversial vibrational assignments from experiment are clarified. Our results clearly show that some structural features like N-methylation, side-chain H-bond, and steric side-chain interactions should also be taken into account when discussing the spectral signatures of secondary structures. These new insights can be very useful for peptide conformation analysis in general.
J. Phys. Chem. A, Vol. 114, No. 36, 2010 9773 Acknowledgment. We gratefully acknowledge financial support from the German Research Council (DFG) through project MA 1356-8/2 and the Collaborative Research Center (SFB) 450. References and Notes (1) Borel, J. F.; Feurer, C.; Sta¨helin, H. U. Agents Actions 1976, 6, 468–475. (2) Rosen, M. K.; Schreiber, S. L. Angew. Chem., Int. Ed. Engl. 1992, 31, 384–400. (3) Pflu¨gl, G.; Kallen, J.; Schirmer, T.; Jansonius, J. N.; Zurini, M. G. M.; Walkinshaw, M. D. Nature 1993, 361, 91–94. (4) Altschuh, D.; Braun, W.; Kallen, J.; Mikol, V.; Spitzfaden, C.; Thierry, L. C.; Vix, O.; Walkinshaw, M. D.; Wuthrich, K. Structure 1994, 2, 963–972. (5) Krarochvil, B; Husak, M; Jegorov, A. Chem. Listy 2001, 95, 9– 17. (6) Kessler, H.; Koock, M.; Wein, T.; Gehrke, M. HelV. Chim. Acta 1990, 73, 1818–1832. (7) Kessler, H.; Loosli, H. R.; Oschkinat, H. HelV. Chim. Acta 1985, 68, 661–681. (8) Loosli, H. R.; Kessler, H.; Oschkinat, H.; Weber, H. P.; Petcher, T. J.; Widmer, A. HelV. Chim. Acta 1985, 68, 682–704. (9) Shaw, R. A.; Mantsch, H. H.; Chowdhry, B. Z. Can. J. Chem. 1993, 71, 1334–1339. (10) Stevenson, C. L.; Tan, M. M.; Lechuga-Ballesteros, D. J. Pharm. Sci. 2003, 92, 1832–1843. (11) Bodack, L. A.; Freedman, T. B.; Chowdhry, B. Z.; Nafie, L. A. Biopolymers 2004, 73, 163–177. (12) Shaw, R. A.; Mantsch, H. H.; Chowdhry, B. Z. Int. J. Biol. Macromol. 1994, 16, 143–148. (13) Dancer, R. J.; Jones, A.; Fairlie, D. P. Aust. J. Chem. 1995, 48, 1835–1841. (14) Bernardi, F.; Gaggelli, E.; Molteni, E.; Porciatti, E.; Valensin, D.; Valensin, G. Biophys. J. 2006, 90, 1350–1361. (15) Bernardi, F.; D’Amelio, N.; Gaggelli, E.; Molteni, E.; Valensin, G. J. Phys. Chem. B 2008, 112, 828–835. (16) Barth, A. Biochim. Biophys. Acta 2007, 1767, 1073–1101. (17) Vass, E.; Hollsi, M.; Besson, F.; Buchet, R. Chem. ReV. 2003, 103, 1917–1954. (18) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.; Vreven, T., Jr.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, J. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, M.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, ReVision C.02; Gaussian, Inc.: Wallingford, CT, 2004. (19) Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652. (20) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1988, 37, 785–789. (21) Vreven, T.; Morokuma, K. J. Comput. Chem. 2000, 21, 1419–1432. (22) Hehre, W. J.; Ditchfield, R.; Pople, J. A. J. Chem. Phys. 1972, 56, 2257–2261. (23) Frisch, M. J.; Pople, J. A.; Binkley, J. S. J. Chem. Phys. 1984, 80, 3265–3269. (24) Collins, J. B.; Schleyer, P. v. R.; Binkley, J. S.; Pople, J. A. J. Chem. Phys. 1976, 64, 5142–5151. (25) Cheeseman, J. R.; Frisch, M. J.; Devlin, F. J.; Stephens, P. J. Chem. Phys. Lett. 1996, 252, 211–220.
JP102206Z
