Revealing the Relationship between Electric Fields and the Conformation of Oxytocin Using Quasi-Static Amide-I Two-Dimensional Infrared Spectra.

It is reported that the cis/trans conformation change of the peptide hormone oxytocin plays an important role in its receptors and activation and the cis conformation does not lead to antagonistic activity. Motivated by recent experiments and theories, the quasi-static amide-I 2D IR spectra of oxytocin are investigated using DFT/B3LYP (D3)/6-31G (d, p) in combination with the isotope labeling method under different electric fields. The theoretical amide-I IR spectra and bond length of the disulfide bond are consistent with the experimental values, which indicates that the theoretical modes are reasonable. Our theoretical results demonstrate that the oxytocin conformation is transformed from the cis conformation to the trans conformation with the change of the direction of the electric field, which is confirmed by the distance of the backbone carbonyl oxygen of Cys6 and Pro7, the Ramachandran plot of Cys6 and Pro7, the dihedral angle of Cβ-S-S-Cβ, and the rmsd of the oxytocin backbone. Moreover, the trans conformation as the result of the turn in the vicinity of Pro7 has a tighter secondary spatial structure than the cis conformation, including stronger hydrogen bonds, longer γ-turn geometry involving five amino acids, and a more stable disulfide bond. Our work provides new insights into the relationship between the conformation, the activation of the peptide hormone oxytocin, and the electric fields.

Introduction

The neurohypophyseal peptide hormone oxytocin (OT) is a cyclic nonapeptide with a disulfide linkage between two cysteine residues at positions 1 and 6. The sequence of the hexacyclic ring is Cys-Tyr-Ile-Gln-Asn-Cys with a free N-terminal amino group. The sequence of the C-terminus tail is Pro-Leu-Gly,[1,2] where the proline (Pro) residue is an essential conformational constraint necessary for the biological activity of OT.[3,4] The structures and functions of OT have been extensively investigated both experimentally and theoretically. OT, which is produced by the hypothalamus and stored and secreted by the posterior pituitary gland,[5] has antiobesity properties,[6−8] associates with labor and lactation,[9−11] and regulates the social behavior in mammals.[12] Larive et al. studied cis/trans conformational equilibrium across the Cys6-Pro7 peptide bond of OT using two-dimensional NMR spectroscopy and found that the interactions between macrocyclic hexapeptide and the tripeptide side chains of OT were unclear and the temperature dependence of the chemical shifts of the resonances for the amide protons was consistent with a higher proportion of structure forms of the hexapeptide rings of the cis isomers.[13] Then, they reported the dynamics of cis/trans isomerization of the Cys6-Pro7 peptide bonds of OT in aqueous and methanol solutions and found that no polar resonance structures were possible in the transition state and the rate of cis/trans interconversion was faster in nonaqueous solvents.[14] Wittelsberger et al. confirmed that cis conformations between Cys6-Pro7 of OT did not lead to antagonistic activity, and a cis/trans conformational change played an important role in OT receptor binding and activation.[15−17] There are several research studies focusing on the spectra of OT, too. Plinska et al. reported and compared the terahertz time-domain spectroscopy investigation on crystalline forms of both OT and derivatives, and the results might be helpful to identify unique biologic signatures of OT.[18] De Barros et al. investigated the Fourier transform infrared information of OT and sustained release using natural rubber latex membranes and identified absorption IR peaks for functional groups of OT.[19] Birech et al. studied the antiobesity influence of OT using Raman spectroscopy and confirmed that OT played an important role in obese rats.[20] Joly et al. studied optical and structural properties of copper–oxytocin dictations in the gas phase, and the experimental and theoretical results showed a global enhancement of the photofragmentation yield as compared to the one recorded for the doubly protonated OT.[21] The QM/MM study on OT electronic properties was conducted by Kitagawa et al. using circular dichroism spectra, and the results demonstrated that the OT electrostatic field at the chiral center was compensated by solvent water.[22] Recently, Liu et al. investigated excited-state spectra of OT, including ultraviolet and fluorescence spectra; the theoretical results agreed well with the experimental ones.[23]

The cis/trans conformations of OT have been widely investigated using 2D NMR spectroscopy.[13−17] Quasi-static amide-I two-dimensional infrared (2D IR) spectra can be viewed as an extended version of 2D NMR spectroscopy,[24,25] which contain lots of detailed information to elucidate the protein three-dimensional structures by the inherent sensitivity to the mechanisms of coupling and energy transfer in intermolecular and intramolecular aspects.[26−30] In proteins, the amide-I vibrational normal mode usually consists of a C=O stretching vibration with weaker contributions from the amide N–H wag. The vibration has a large transition dipole moment, leading to strong IR activity, and is delocalized over many residues, which gives a detailed structural fingerprint of the protein backbone.[31−33] Quasi-static amide-I 2D IR spectra in combination with isotope labeling methods have been extensively used to identify misfolded proteins and get access to the mechanism of the process.[24,34−36]

Structure–activity studies concern the structural features leading to agonism or antagonism of the OT effect on uterus prompted based on the cis/trans conformation,[10,12−17] and it is reported that electric fields of specific intensity and frequency can excite certain vibrational modes of proteins, which alters their conformations.[37−41] Therefore, in this paper, we theoretically study the response of the OT conformation to electric fields using quasi-static amide-I 2D IR spectra in combination with isotope labeling methods. On one hand, we aim to study hydrogen bonds among different residues because OT has an obvious β-turn geometry.[2−4,12−14,16,42] On the other hand, based on the Cys6-Pro7 isomer, the cis/trans conformations of OT are detected using 2D IR spectra under different electric fields.[13−17]

Experimental and Computational Methods

Experimental Methods

The OT crystal sample, produced by Jiangsu Aikon Biopharmaceutical R&D Co., Ltd, China, of about 2 mg is well mixed with potassium bromide (KBr) of about 20 g, and the mixtures are pressed into thin slices at 15 Mpa pressure for 2 min; then, infrared light is used to dry the slices for about 30 min. Finally, an infrared spectrometer, which has a wavelength accuracy of 0.01 cm–1 and a wavelength resolution of 0.5 cm–1, is used to measure the infrared spectrum among 400–4000 cm–1.

Computational Methods

The initial geometry of an OT molecule is taken from the X-ray structure (PDB ID: 1xy2,[43]Figure ). The replacement of Cys1 with Mpr1, which is a nonprotein residue and is less bulky than Cys1, has been shown to affect the chemical environment and consequently the nature of the disulfide bond linking residues 1 and 6,[16,22] and the backbone carboxyl groups of Cys6 and Pro7 are labeled with 13C=18O, which has about a 60 cm–1 red shift.[24,34−36] The geometry optimization and anharmonic frequency calculation of OT are carried out using the B3LYP (D3) functional and 6-31G (d, p) basis set as implemented in the Gaussian 16 program,[44−49] where the SMD continuum water model is included.[50] The static electric fields along the X-axis (Figure , red arrow) are in the range of −1.028–1.028 V/nm, which have been extensively investigated.[37−39,51,52] The optimized geometries are characterized as the true localized minimum energy on the potential energy surface without imaginary frequency based on the Gaussian 16 default convergence criterion. With the help of the vibration energy distribution analysis (VEDA) program,[53] the anharmonic frequencies are assigned as normal vibration models.

Figure 1

OT molecular geometry. Carbon (C), oxygen (O), nitrogen (N), sulfur (S), and hydrogen atoms are represented by balls of blue green, red, blue, yellow, and white colors, respectively. The colored ribbons represent different residues.

The calculation of quasi-static amide-I 2D IR spectra has been described in detail elsewhere.[24,28,40] In summary, two-exciton Hamiltons are diagonalized, which are constructed from the ground-state, two-excition, and combination band frequencies of amide-I, and the anharmonicity which is necessary to create 2D IR spectra is calculated with anharmonic frequency.[49] The transition dipole moment vectors between the vibrational ground state and first excited state for each of the normal modes are obtained from the DFT calculation. In order to obtain the transition dipole moment vector between the one-exciton to two-exciton states, harmonic approximation is employed, that is, the corresponding ground to one-exciton state transition dipole moment vector is multiplied by .[24] In general, the fourth power of the transition dipole moments involved is proportional to the 2D IR peak intensity in a given vibrational mode.[24,54] Furthermore, the waiting time Tw is set to 3 ps.

Results and Discussion

The ability of an IR probe to quantitatively report on the local electric field is crucial for successful application to study the local structure in proteins, and numerous IR spectroscopic studies have been reported for the OT molecule.[18−21] Thus, for the sake of direct comparison, the IR spectra of the OT molecule are studied experimentally and theoretically (Figure ). The theoretical anharmonic IR peak of the disulfide bond lies on 552 cm–1, which is slightly larger than our experimental value of 547 cm–1 and other experimental values of 542 cm–1,[19] and the strength of the IR peak of the disulfide bond is much smaller than that of amide-I. The theoretical length of the disulfide bond is 2.095 Å, which is consistent with another theoretical value of 2.096 Å[22] and slightly larger than the experimental value of 2.035 Å.[22] The theoretical anharmonic IR peaks of amide-I of the isotope-labeled Cys6 and Pro7 are 1587 and 1615 cm–1, respectively, and the theoretical anharmonic IR peaks of amide-I of other residues are 1651, 1676—, and 1711 cm–1, which are consistent with our experimental range of 1655–1675 cm–1 and Barros group experimental range of 1643–1658 cm–1.[19] In conclusion, the theoretical anharmonic amide-I IR spectra agree well with the experimental ones, and the theoretical models are reasonable.

Figure 2

Experimental and theoretical IR spectra of the OT molecule. The theoretical anharmonic IR spectra only include amide-I of the OT molecule.

In what follows, we present normalized quasi-static amide-I 2D IR spectra of isotope-labeled Cys6 and Pro7 under different electric fields (Figure ), whose spectra are interpreted in the context of the mode assignments, frequencies, and relative intensities. The diagonal peaks of the 2D IR spectra reflect the corresponding 1D spectra, and the intensities of 2D IR spectra are scaled with the transition dipole as μ4, which is confirmed in the top panel and bottom panel of Figure .[24,28,32] According to vibration energy distribution,[54] the anharmonic amide-I frequencies of isotope-labeled Cys6 are lower than those of isotope-labeled Pro7, and the frequencies both are red-shifted when the electric fields are applied. The red-shifted phenomenon indicates that the electric fields have an important impact on the electrostatic environment and transition coupling of isotope-labeled Cys6 and Pro7.[55] From Figure , the off-diagonal anharmonicity Δ76 is about half the diagonal anharmonicity Δ6, but the off-diagonal anharmonicity Δ76 is zero, that is, one cross-peak disappeared when the electric field is 0.5142 V/nm (Figure d), which indicates that there is a weaker coupling between isotope-labeled Cys6 and Pro7. Usually cross-peaks in 2D IR spectra are caused by coupling between vibration modes and the reasons are as follows: It is reasonable to consider the one-exciton Hamiltonian for linear spectroscopy in a coupled dimer[24,28,32]where β and ω represent the coupling constant and frequency, respectively. The exciton eigenvalues are

Figure 3

Normalized and simulated quasi-static amide-I 2D IR spectra and the corresponding IR spectra (top panel) of isotope-labeled Cys6 and Pro7 under different electric fields (V/nm). E_Field (a) −1.028; (b) −0.5142; (c) 0; (d) 0.5142; and (e) 1.028. The residue labels represent the corresponding amide-I modes.

The eigenstates of E1 and E2 arewhere α represents the mixing angle

When the coupling constant is small compared to the frequency splitting that is

Their mixing angle α is small, and the exciton states will be localized on the individual sites.

Then, the coupling constants β12 can be calculated based on perturbation theory[56]where χ represents the positive constant and Δ12 and Δ2 represent the off-diagonal anharmonicity and diagonal anharmonicity, respectively. According to eq , the coupling constants are calculated (the detailed information is given in the Supporting Information Table S1), and the results are presented (Table ). When the electric fields are antiparallel to the X-axis, the coupling constants are larger than those under other electric fields. Especially, the coupling constant is zero when the electric field is 0.5142 V/nm. Because the coupling constants are inversely proportional to the distance (r) between two local modes, that is, ,[24,28,32] the backbone of isotope-labeled Cys6 and Pro7 has undergone major changes when the electric fields are antiparallel to the X-axis. The distance of the carboxyl oxygen (O) atom of the backbone between isotope-labeled Cys6 and Pro7, the rmsd of the OT backbone, and the Ramachandran diagram of Cys6 and Pro7 are studied,[57] and the results are shown in Table and Figure . When the electric fields are antiparallel to the X-axis, the distances of O6–O7 are about 3.5 Å, which are smaller than the distances, about 4.2 Å, of O6–O7 under other electric fields (Table ), which indicates that the geometry of the backbone of isotope-labeled Cys6 and Pro7 varies greatly. The root mean squared deviation (rmsd) of the protein backbone usually reflects the degree of deviation between two protein structures, and we calculated the rmsd of the OT backbone (Table ). When the electric fields are antiparallel to the X-axis, the value of rmsd is about 1.387 Å, which is larger than that while the electric fields are parallel to the X-axis. The change of the rmsd indicates that the OT conformation changes greatly while the electric fields are antiparallel to the X-axis. What is more is that when the electric fields are antiparallel to the X-axis, the (φ, ψ) values of isotope-labeled Cys6 and Pro7 are approximately (−172°, 145°) and (−55°, −33°), respectively, which are obviously different from those under other electric fields (Figure ). The Ramachandran diagram shows that the orientation of the backbone of isotope-labeled Cys6 and Pro7 has changed a lot, too. After careful differentiation, the conformation of OT belongs to the trans conformation, while the electric fields are antiparallel to the X-axis, but the conformation of OT is the cis conformation, while the electric fields are parallel to the X-axis.[13,14] Our results conclude that the electric field plays an important role in the trans/cis conformation of OT, and the OT conformation is transformed from the cis conformation to the trans conformation with the change of the direction of the electric field.

Table 1

Coupling Constants (β), Distance (Å) of the Backbone Oxygen between Isotope-Labeled Cys6 and Pro7, and rmsd (Å) of the OT Backbone under Different Electric Fields (V/nm)

E_field	β76	dO6–O7	rmsd
–1.028	28.3χ	3.460	1.3871
–0.5142	31.2χ	3.499	1.3870
0	17.4χ	4.149	0
0.5142	0	4.276	0.1358
1.028	19.0χ	4.255	0.7255

Figure 4

Ramachandran diagram of isotope-labeled Cys6 and Pro7 under different electric fields.

As described above, the OT molecule has a γ-turn geometry[13−17] and the 2D IR spectra have unique advantages in detecting vibrational coupling and energy transfer among intramolecular and intermolecular reactions.[24,28,32] Therefore, in the section, we use quasi-static amide-I 2D IR spectra (Figure ) to study the interaction among the hexacyclic peptide of OT. The 2D IR spectra are complicated, and the diagonal peaks of 2D IR spectra usually contain several vibration modes from different backbone carboxyl groups stretching based on vibration energy distribution.[53] The diagonal anharmonicities of the mode including Asn5 are positive, but the diagonal anharmonicities of the Tyr2 mode are negative (Figure a,b,d), which indicates that the first excited energy is larger than the second excited energy.[24,39] What is more is that the cross-peaks reveal the complex interaction among hexacyclic peptides including hydrogen bonds and van der Waals force. Spyranti et al. investigated the hydrogen bonds in OT and deamino OT using NMR spectroscopy and found the hydrogen bonds between Tyr2 and Asn5.[13−17] Therefore, the revealing noncovalent interaction method (NCI)[58−60] and the core–valence bifurcation index (CVB index) are used to study the hydrogen and the corresponding strength,[61−63] respectively (Figure ). The CVB index is defined aswhere ELF is the abbreviation for the electron localization function and D and A are the abbreviation for the hydrogen bond donor and acceptor, respectively. ELF(C–V) represents the value of the bifurcation point between the core and valence, and ELF (DH–A) represents the value of the bifurcation point between the core and valence of hydrogen atoms and acceptors. Generally, the smaller CVB index indicates the higher hydrogen bond strength. Specifically, all geometries include the hydrogen bond involving Tyr2 and Asn5, but the strength is different. When the electric field is −1.028 V/nm, the CVB index between Tyr2-CO and Ans5-NH is 0.04769, which is the smallest. When the electric field is −0.5142 V/nm, the CVB index between Tyr2-NH and Ans5-CO is 0.06051, which is the largest. The abovementioned data suggest that the OT molecules include regular γ-turns. What is more is that when the electric fields are antiparallel to the X-axis, the crystal structure of OT shows a turn in the vicinity of Pro7 (Figure a,b), and the turn is characterized by two new strong hydrogen bonds between Cys6-CO and Gly9-NH and between Gly9-CO and Gln4-NH, where the CVB index (about 0.0492) between Cys6-CO and Gly9-NH is larger than that between Gly9-CO and Gln4-NH. We describe the turn with the distance between the backbone oxygen atoms of Cys6 and Pro7, and Table clearly indicates the existence of the abovementioned trans states. When the electric fields are parallel to the X-axis (Figure d,e), the shapes of the C-terminus tail are far away from the hexacyclic peptides. As one would expect, a weak hydrogen bond is identified between the carbonyl oxygen of the Asn5 side chain and the methylene hydrogen of the Pro7 side chain, while the electric field is 1.028 V/nm, whose CVB index is 0.07086. In conclusion, when the electric fields are antiparallel to the X-axis, the OT molecules have longer γ-turns including five amino acids and stronger hydrogen bonds between Tyr2 and Asn5, indicating the tighter secondary spatial structure of the OT molecules.

Figure 5

Normalized and simulated quasi-static amide-I 2D IR spectra and corresponding IR spectra (top panel) of hexacyclic peptide under different electric fields (V/nm). E_Field (a) −1.028; (b) −0.5142; (c) 0; (d) 0.5142; and (e) 1.028. The residue labels represent the corresponding amide-I modes.

Figure 6

Hydrogen bonds among residues under different electric fields (V/nm). The purple isosurface represents the 0.75 a.u. region, and the numbers represent the CVB index. E_Field (a) −1.028; (b) −0.5142; (c) 0; (d) 0.5142; and (e) 1.028.

The different residues in position 1 play an important role in the nature of the disulfide bond which is between Mpr1 and Cys6,[16,43] and its stability is studied under electric fields based on Mayer bond order density (BOD).[58,64] The nature of Mayer BOD corresponds to the delocalization index in the Hibert atomic space, which is more comprehensive for investigating the stability of covalent bonds, and the number of Mayer BOD is favorable to the stability of covalent bonds. The values of Mayer BOD are close to 1, which indicates that the disulfide bond is the single covalent bond (Figure ). When the direction of the electric field is antiparallel to the X-axis, the value of Mayer BOD is about 0.993. When the electric field is 1.028 V/nm, the value of Mayer BOD is 0.940, which is the smallest. The data conclude that when the electric fields are antiparallel to the X-axis, the OT molecules contain a more stable disulfide bond. What is more is that, the dihedral angles of Cβ-S-S-Cβ (Figure ) are calculated under different electric fields, and the results are shown in Table . When the electric fields are antiparallel to the X-axis, the dihedral angels are about 84°. However, the dihedral angles are obviously decreased to 60° when the electric fields are parallel to the X-axis. The change of the dihedral angle indicates that the electric fields have changed the chemical surroundings of Cys6, and the OT conformation has changed a lot.

Figure 7

Values of Mayer BOD of the disulfide bond under different electric fields.

Table 2

Dihedral Angle (degree) of Cβ-S-S-Cβ under Different Electric Fields (V/nm)

E_field	–1.028	–0.5142	0	0.5142	1.028
Cβ-S-S-Cβ	84.3	83.5	60.9	59.8	57.3

Conclusions

We present the experimental infrared spectra and the theoretical quasi-static amide-I 2D IR spectra of the peptide hormone oxytocin using DFT/B3LYP (D3)/6-31G (d, p) in combination with the isotope labeling method under different electric fields. When the oxytocin is free of the electric field perturbation, the theoretical anharmonic amide-I IR spectra agree well with our experimental IR spectra, and the bond length of the disulfide bond agrees well with the experimental values. By comparison of amide-I 2D IR spectra of isotope-labeled Cys6 and Pro7 under different electric fields, the coupling constants between them are calculated, and the results demonstrate that the oxytocin conformation changes a lot when the electric field changes the direction, which is confirmed by the distance of the backbone carbonyl oxygen of isotope-labeled Cys6 and Pro7, the Ramachandran plot of isotope-labeled Cys6 and Pro7, the dihedral angle of Cβ-S-S-Cβ, and the rmsd of the OT backbone. After careful differentiation, the conformation of OT belongs to the trans conformation as the result of the turn in the vicinity of Pro7 while the electric fields are antiparallel to the X-axis, but the conformation of OT is the cis conformation while the electric fields are parallel to the X-axis. Through the revealing noncovalent interaction method and comparing the core–valence bifurcation index, we find that the trans conformation has a tighter secondary spatial structure than the cis conformation, including stronger hydrogen bonds and longer γ-turn geometry involving five amino acids. Last but not least, the disulfide bond is more stable in the trans conformation than in the cis conformation based on Mayer bond order density.