Conformational Properties of Oxytocin in Dimethyl Sulfoxide Solution: NMR and Restrained Molecular Dynamics Studies R. BHASKARAN,* 11-CHIN CHUANG, and CHIN YUt

Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China


The conformation of oxytocin, the neurohypophyseal nonapeptide hormone, in solution in deuterated dimethyl sulfoxide has been determined by 'H-nmr. The structural determination is based on the experimental data set of nuclear Overhauser effect restraints. Obtained after the restrained molecular dynamics simulation on an initial structure of extended conformation, five resultant structures satisfy the experimental restraints well. These structures resemble that of the crystal structure of deamino-oxytocin, an analogue of oxytocin, in terms of a close correlation observed both at two 0-turn regions of the 20-membered tocin ring and at the tripeptide tail end. Based on this comparison and analysis of restrained molecular dynamics trajectories, we found that, although the turns are stabilized by the formation of hydrogen bonds, the oxytocin molecule possesses a slight twist in DMSO solution relative to the orientation of deamino-oxytocin in the crystalline state. Analyses of oxytocin conformation indicate that the tripeptide tail is more flexible than the tocin ring. 0 1992 John Wiley & Sons, Inc.

INTRODUCTION The neurohypophyseal hormone, oxytocin, has important biological properties. It causes ejection of milk from mammary tissue and uterine contraction, and has an antidiuretic function.lS2It also binds in viuo to a group of intracellular carrier proteins known as neurophy~ins.~ Oxytocin has the following primary structure (numbers indicate the position of individual amino acid residues) : Cysl -Tyr2-Ile3-Gln4-Asn5-Cys6L I Pro7-Leu8-GlyS (NH2)

It consists of a 20-membered tocin ring (from Cysl to Cys6) and an acyclic tripeptide tail (from Pro7 to Gly9). The cyclic structure is completed by the Biopolymers, Vol. 32, 1599-1608 (1992) 0 1992 John Wiley & Sons, Inc.

CCC 0006-3525/92/121599-10

* On leave from the Department of Physics, Bharathidasan University, Tiruchirapalli, India. ' T o whom correspondence should be addressed.

formation of a disulfide bond between Cysl and Cys6 ( marked above ) . In order to explain the interaction of the oxytocinuterine receptor, diverse models have been proposed for the three-dimensional (3D) structure of oxytocin, on the basis of energy m i n i m i ~ a t i o n , x-ray ~,~ d i f f r a ~ t i o n ,and ~ ' ~ CD,8-11Raman,8,12and nmr9,13-24 spectroscopic techniques. These models reveal that the conformation of oxytocin consists of two p-turns; one in the cyclic moiety involves the sequence Tyr2113-Gln4-Asn5, and the second, a hairpin turn, involves the C-terminal sequence CysG-Pro7-Leu8G l ~ g . ' ~ ,According '~ to the cooperative model of Walter et al., the lipophilic side chains of residues Ile3, Gln4, Pro7, and Leu8, which mainly determine the formation of the two reverse turns and of which each is at the corner of the reverse turn, make possible the binding of oxytocin to a receptor; the positions of Tyr2 and Asn5 are considered critical for the biological r e s p o n ~ e . ' In ~,~ contrast, ~ the dynamic model emphasizes the conformational flexibilities of the acyclic tripeptide and its relationship with the 20-membered cyclic ring to be critical for binding.16,18,20 A omplete knowledge of the molecular 1599



conformation is an utmost need to understand the biological function of the hormone. At the present stage, determination of the detailed structure, being incomplete, remains to be solved. The availability of the crystal structure of deamino-oxyt~cin,~’~ an analogue of oxytqcin, provides supporting data to compare with the structure of oxytocin in solution. Two-dimensional (2D) nmr25 and restrained molecular dynamics 26-28 ( RMD ) techniques have been combined to determine the 3D molecular structure in solution. From the nuclear Overhauser enhancement (NOE) data, one derives a set of distance restraints. An initial structure is subsequently optimized and refined using the RMD method, to obtain energetically stable structures with minimum distance violations from the conformational space. We found that the solution conformation of oxytocin is similar to that of the x-ray structure of deaminooxytocin. Our objectives in this work have been to determine the conformation of oxytocin in DMSO solution, to compare this structure with that of its analogue, and to characterize the dynamical nature of conformational states.

in which dlm denotes a known calibrated distance that is considered to be 1.78 A for geminal protons; qmand aij denote the cross relaxation rates for spins l,m and i, j respectively. We used the initial slopes of the NOE buildup curves for this purpose. Computational Methods

To determine the 3D structure, we used the RMD simulation by translating the NOE interproton distances. All computations were done on Silicon Graphics 4D/25G and microVAX 111 computers. The energy minimization and RMD calculations were performed with the program CHARMM.32The molecular graphics software QUANTA (Version 3.2.3, Polygen Corporation, 200 Fifth Avenue, Waltham, MA) was used for generation, display, analysis, and plotting of the molecular structures. Restrained Molecular Dynamics

For calculations of molecular dynamics trajectories, the empirical energy function was used, which includes harmonic potential-energy terms for bond MATERIALS A N D METHODS lengths, bond angles, dihedral angles, and functions for van der Waals, electrostatic interactions. The Experimental cutoff radius for the nonbonded interaction was set at 14 A in order to include all significant interactions. Oxytocin (Sigma, 10 mg) was dissolved in DMSOSolvent molecules were not explicitly introduced in (Aldrich, 500 p L ) to make a solution of concentration 20 mmol L-l. 4,4-Dimethyl-4-silapentane- the computations; instead their presence was simulated by the inclusion of a distant-dependent di2-sulfonate was used as internal standard. The electric c ~ n s t a n t .Furthermore, ~~’~~ we added a disnmr tube was degased and sealed. Phase-sensitive tance-restraint function to the energy terms to inNOE spectroscopy (NOESY)29 spectra with five clude the NOE distance information. mixing periods, viz., 30, 60, 90: 120 and 300 Calculations of dynamics simulation were perms, were recorded on a 400-MHz spectrometer formed by means of the Verlet algorithm.35The in(Bruker AM-400) equipped with an Aspect 3000 terval for the integration was 1fs. During the heating computer. Quadrature detections in F1were achieved and equilibration periods, the velocities of the atoms using a time proportional-phase-incrementation were reassigned to the appropriate temperatures evapproach3’; 512 tl increments were recorded, with 2048 complex data points. For each free induction, ery 0.1 ps. the decays were Fourier transformed on F2using a Kaiser window and on F1 using a 45O phase shifted RESULTS AND DISCUSSION sine-bell window. All 2D data were processed on a microVAX 111 computer, using the FTNMR softExtraction of NMR Parameters ware kindly provided by Dr. D. Hare. The ‘H-nmr resonances for oxytocin have been preThe interproton distance information was derived viously These results indicate that by means of the NOE data from the five NOESY this molecule possesses two P-turns covering resispectra. First, the cross peaks of these spectra were dues Tyr2 to Asn5 and Cys6 to Gly9, respectively, integrated by volume; then the NOE buildup curves with hydrogen bonding between the i and ( i 3 ) were plotted after least-squares fitting. The interresidues in the peptide backbone. The NOESY proton distances dij were calculated according to the relation 31 spectra were particularly helpful in identifying the



conformation of oxytocin. A contour plot of the NOESY spectrum with a mixing period 120 ms observed for the amide proton range on F2appears in Figure 1. All clearly observed 97 NOE cross peaks were used to set up distance constraints so as to define the conformation of the oxytocin. The crossrelaxation rates for each proton pair of NOE were



-. O






Structure Refinement by RMD



@ I







-0. 0






,4,1 8.0


obtained from NOESY spectra recorded with five mixing periods and the interproton distances were calculated as described above. As the NOE-derived interproton distances may have uncertainties relative to the actual distances due to experimental error, a correction factor was added for the upper and lower bounds. Because of rotation of methyl groups, flipping of aromatic rings, and inability to make stereospecific assignments, pseudo-atom correction factors were applied. The maximum possible error due to the introduction of pseudo atoms was incorporated with the constraints by additional correction terms as described by Wuethrich et a1.38 In addition, it is expected that the measurement error is inherent in the calculation of NOE distances of the tripeptide tail region due to its flexible nature. Hence, the values obtained are considered to be from its averaged state in solution.




a- H


PPm Figure 1. NOESY spectrum of oxytocin for a mixing time of 120 ms. Only amide proton region is shown on x axis.

The initial structure for the RMD calculation has been generated by the computer model incorporating the extended chain conformation. All amide bonds were fixed to be trans and the side chains fixed in their extended states. The disulfide patching was applied to close the structure to a ring form. Using the interproton distance constraints for RMD simulation, we took explicitly into consideration all hydrogen or pseudo atoms to which the NOE constraints are referred. One hundred fifty-four distance constraints were applied in total. The constraints on interproton distances used for the RMD run on oxytocin are listed in Table I. No explicit hydrogenbonding terms or hydrogen-bonding constraints were used in the present simulation. The molecule was allowed to relax first by unconstrained energy minimization for 500 steps of the conjugate gradient method,39to remove strains caused on definition of the interchain bonds. We initiated the RMD simulation by assigning velocities to all peptide atoms, randomly selected from a Maxwellian distribution. The molecule was first heated gradually from 0 to 300 K with velocities reassigned every 0.1 ps from the distribution at temperatures incremented by 50 K at each reassignment. The initial heating steps were selected for a smaller drift in the temperature variation to increase the temperature to the required limit of 300 K. Following this initial heating procedure, after the temperature of the molecule reached 300 K, it was then equilibrated to the same number of steps as was carried out for heating, to become relaxed at the same temperature. At this stage, the atomic velocities were



Table I Input Distance Constraints for RMD Simulations on Oxytocin’

Table I (Continued) Proton Pair

Proton Pair Atom 1 Cysl






Distance Constraints Atom 2

Cysl 5 r 2 Cys6 Tyr2 Tyr2 Ile3 Cys6 Asn5 Tyr2 Tyr2 Ile3 Am5 Tyr2 Ile3 Asn5 Asn5 Ile3 Ile3 Ile3 Ile3 Ile3 Gln4 Ile3 Ile3 Gln4 Ile3 Ile3 Gln4 Asn5 Cys6 Gln4 Gln4 Asn5 Gln4 Asn5 Cys6 Gln4 Cys6 Asn5 Asn5 Cys6 Asn5 Cys6 Pro7 Asn5 Cys6 Gly9 Cys6 Cys6 Pro7 Cys6 Pro7

(A) 2.58-3.78 1.65-2.25 1.95-3.55 2.75-3.35 1.18-3.01 2.60-3.20 2.00-4.50 2.00-6.00 2.12-3.32 3.19-6.19 2.12-2.72 2.00-6.00 2.75-6.35 2.67-4.12 2.00-6.00 2.00-8.40 2.35-2.95 2.00-5.60 2.34-2.94 3.02-3.62 3.06-4.33 1.80-2.40 2.45-3.65 3.07-4.27 2.00-4.50 2.00-6.00 2.00-6.00 2.03-2.63 2.77-3.37 2.00-6.00 2.28-3.49 2.23-3.52 2.16-2.76 2.21-4.01 3.09-4.29 2.00-6.00 2.47-4.27 2.00-6.00 2.38-2.98 2.26-3.46 2.42-3.02 1.77-2.97 1.97-2.57 2.00-6.00 2.06-3.86 2.68-3.88 2.00-6.00 2.34-2.94 2.36-3.86 2.67-4.38 2.21-3.48 2.10-3.47



mn i q mn iq m mn mq i i i i i mn mn

mn i i ii m mn

mn i i

mn ii m mn i m i m

Distance Constraints Atom 2

Atom 1 Leu8 Gly9 Pro7 Leu8 Leu8 Pro7 Leu8 Leu8 Leu8 Gly9 Gly9 Gly9

(A) 2.00-6.00 2.00-6.00 2.10-3.48 1.SO-2.40 1.94-4.14 2.00-6.20 2.51-4.01 2.00-5.60 2.20-3.40 2.22-3.22 2.00-6.00 2.85-4.05

mn mn i m ii i i

mn i

a Lower and upper bounds are given for each constraints. The letters “i,” “m,” and “q” after the upper distance bounds indicate the correction factors 0.6,l.Oand 2.4 8, added to the corresponding upper bounds of the constraints respectively; n refers to the cases whose cross-peak volumes are too small to be integrated in NOESY spectra with mixing time less than 200 ms, for which an upper limit of 6 A was used.

scaled by an appropriate factor to keep the temperature constant. The trajectories were then continued for a subsequent period of 20 ps simulation. During the course of these simulation steps, the NOE energy was observed to decrease from its initial value (because of its extended conformation ). This decrease in the energy makes the extended peptide atoms approach each other to enter into the secondary structural state and then finally converge to attain their 3D structure. In each stage, the experimental NOE constraints applied for the RMD run were properly obeyed. The variation of the NOE energy with respect to dynamics intervals is shown in Figure 2. Thereby, we understand that the final 12 ps simulation structures possess a smaller variation in their NOE energies between any two structures generated consecutively. This result indicates that in these regions the conformations attained are of comparable energy to each other, after strictly following the experimental NOE constraints. Thus in these regions the structures have fewer NOE constraint violations relative to the initial and other (previous) structures generated during the RMD run. Selection and Analysis of Calculated Structures

Like the variation in the NOE energy plot (Figure 2 ) , the potential-energy trace indicated a region of



tions of oxytocin in solution indicating its flexible character, 16~18,20wobbling around its mean conformation. \

Convergence of Selected Structures

I 1 1 T I 1 ' q - T














Time I P S )

Figure 2. The plot showing the variation of the NOE energy with respect to the RMD time steps in ps. The x axis has been scaled differently in order to cover the whole range of the RMD run.

constant variation in the last 12 ps range (not shown). Despite the observation of an energy range with only slight variation, several local minima are discernible. Of these, we selected five local minima based on their small contribution to the NOE energy and small amount of NOE violation from the experimental constraints. These structures possess hydrogen bonding in the 20-membered tocin ring and in the region of the tripeptide tail. The atoms involved in these two hydrogen bonds are the carbony1 0 of Tyr2 and Cys6 with the amide protons of Asn5 and Gly9, respectively. Thus, the results of the hydrogen-bond participation of the amide protons of Asn5 and Gly9 (proposed to participate in the tocin ring and tripeptide tail end turn regions respectively) agree well with the earlier nmr studies on the temperature dependencies of the amide proton chemical The analysis of secondary structure also yielded the result that all selected structures possess two P-turn conformations, covering the residues Tyr2-Ile3-Gln4-Asn5 and Cys6Pro7-Leu8-Gly9, respectively. These observations agree with published proposals for oxytocin; hence the selected structures represent the conformation of oxytocin in DMSO s o l ~ t i o n . ~ , ' ~ - ' ~ ~ ~ ~ When we analyzed other local minima, we found that these structures showed some agreement with the selected RMD structures. However, they did not possess turns of the same kind we observed. With a slightly different conformation, the hydrogen bond was formed between Tyr2 C=O to Cys6 NH. At the tail region they show a folded conformation with a hydrogen bond between the Cys6 C = O to Leu8 NH. These observations imply that the RMD simulated structures span a closer range of conforma-

The agreement between all selected RMD structures with those of the earlier models indicates the similarity of all the structures. Hence they were superimposed for the best fit of the backbone atoms (N,Ca, C', and 0) of all the residues. The superimposed structures are shown in Figure 3 ( a ) , in which we see that the overall fold of the backbone remains almost the same except for some ill fit at the C' position of Gln4 and the C* position of Pro7. As the tail end experiences some flexibility, the residue joining the tocin ring and the tripeptide tail region (Pro']) shows some ill fit in the superposition of all the residues. Otherwise the two turn regions overlapped well. To emphasize the goodness of overlap, we compared the energy terms including that of the NOE energy (Table 11) for all five selected RMD structures. All energy values are found to be similar and the total energy lies within the narrow range of 1.5 kcal/mole. The greater value of the total energy results from the inclusion of the distant-dependent dielectric constant for the solvent effect. The smaller range of variation in energy terms indicates that these RMD structures represent the solution structure, based on the experimental NOE. According to previous studies, the oxytocin molecule in DMSO has the same conformation as that of the deamino-oxytocin in DMS0.5-7,'3Hence, we compared the similarities of the RMD structures of oxytocin with the crystal structure of its analogue, deamino-oxytocin. The x-ray structure was superimposed on the RMD structures for the best fit of the backbone atoms of all residues. The backbone fold of all structures coincides well; the RMD structures are found to be similar to that of deaminooxytocin. Although the regions of secondary structure fit well, we observed that the orientation of the two @turns of the RMD structures differ slightly from that in the crystal structure, when the molecule was considered as a whole. As the orientation of the P-turn in the tail region is on a different plane from that in the tocin ring region (the former is oriented away from the ring), the superimposed plot does not give a clear view of the tail residues. Hence for clarity, we show two plots individually, one for the tocin ring and the other for the tripeptide tail region. In the former case, the tocin ring backbone atoms were superimposed for



Table I1 Energy Terms for the Five Selected RMD Structures of Oxytocin RMD Structures (kcal/mol) Energy


Torsion 37.79 van der Waals -2.37 Bond length 56.58 Bond angle 91.07 Electrostatic -0.90 NOE constraints 49.59 Total 396.45





40.47 33.88 36.95 32.59 -7.11 -15.97 -12.00 -14.77 46.99 54.74 47.22 53.43 103.50 88.38 103.45 98.40 -0.74 -0.82 -0.75 -0.89 49.50 59.04 50.89 54.72 396.80 395.83 395.37 395.91

the best fit of the RMSD values (Figure 3b). Similarly, the tail region was superimposed for the best fit of RMSD values among the residues Cys6 to Gly9 (Figure 3c); the x-ray structure is indicated by the thick line and the RMD structures by thin lines. Thus Figure 3 ( b ) and ( c ) clearly reveals the similarities of the solution conformations of oxytocin with that of the crystal structure of deamino-oxytocin. This result is consistent with the proposed models.5,15,18,20 From the close convergence observed in Figure 3 (a-c) , we understand that the solution structure of oxytocin closely resembles the x-ray crystal structure of deamino-oxytocin except for ti little difference in the orientation of the two P-turns in the molecules. As the second p-turn is in the tail region, it spans a narrow range of conformations during the RMD run. Characteristics of Converged Structures

Figure 3. ( a ) Best fit superposition of the backbone atoms (N, C", C', and 0) of the five selected RMD structures of oxytocin. The C" atom positions are numbered. ( b ) Superposition of the first six residues (tocin ring) of the RMD structures of oxytocin (thin lines), and the crystal structure of deamino-oxytocin (thick line). Only the backbone atoms are shown and the C" atom positions are numbered. ( c ) Superposition of the @-turninvolving the tripeptide tail end residues for the structures shown in ( b ) .

From the superposed plots [Figure 3(a-c)], the overall shape of the oxytocin fold are well reproduced, compared with that of the deamino-oxytocin. The backbone dihedral angles of the five converged structures are compared with that of the crystal structure of deamino-oxytocin (Table 111). The contents reveal some differences in the dihedral angles of 4 for the residues Ile3, Asn5, and Cys6, and 1c/ values for Pro7. This effect confirms our observation that the solution structure of oxytocin possesses slightly twisted ,&turns ( oriented differently) relative to the x-ray structure. In order to confirm the dihedral angles 4 obtained from RMD simulation with the nmr results, we computed the dihedral angles 4 from the coupling constants J(NH,Ha) reported earlier.16 The Karplus-type equation given by Bystrov is used to calculate the dihedral angles.40Here 0 is the angle be-



Table I11 Backbone Dihedral Angles of the Five Selected RMD Structures of Oxytocin with Those of the X-Ray Structure of Deamino-Oxytocin RMD Structures Residue

Dihedral Angle

Cysl Tyr2 Tyr2 Ile3 Ile3 Gln4 Gln4 Am5 Asn5 Cys6 Cys6 Pro7 Pro7 Leu8 Leu8 Gly9

d d

* * * *d * * * d

4 4




X-Ray Structure






62 -123 165 -54 123 52 28 -147 76 -146 94 -73 -2 -85 -35 168

125 -179 153 60 84 74 -2 61 70 37 73 -74 74 -138 -59 -163

89 -153 167 52 89 72 -17 67 60 57 97 -90 37 -138 39 -168

134 -170 151 55 79 89 -19 82 74 49 79 -77 75 -146 75 -99

73 -153 167 60 82 56 1 75 38 76 88 -50 77 -158 77 -102

72 -148 177 54 84 64 5 39 70 56 92 -46 74 -168 74 -62

tween the projection of the bonds NH and C"H on a plane perpendicular to the N-C" axis and is related to 4 by 0 = 160 - 41. Table IV summarizes the coupling constants for the residues Tyr2 to Gly9 and indicates their possible values of 0 and 4. A comparison of these dihedral angles with those of the values observed for the five selected RMD structures reveals a close agreement (especially in the region of tocin ring) duly confirming the results of RMD calculation. To obtain the degree of closeness of the converged structures with the x-ray structure, we computed the atomic root mean square deviation (RMSD) values between the five converged RMD structures and the x-ray structure (Table V). The values indicated refer to the backbone atoms (N, C ",C', and 0 ) only. The values in the lower triangle correspond to all residues whereas those in the upper triangle Table IV Dihedral Angles Derived from Coupling Constants, J(NH, H")

Tyr2 cys3 Gln4 Asn5 Cys6 Leu8 Gly9

7.0 5.0 6.0 6.0 7.5 7.5 6.0

20,145 38,132 30,138 30,138 14,148 14,148 30,138

40,80, -85, 22,98, -72, 30,90, -78, 30,90, -78, 46,74, -88, 46,74, -88, 30,90, -78,

-155 -168 -162 -162 -152 -152 -162

correspond to the ring residues. Thus the RMSD values of the backbone atoms of all residues among the five RMD structures vary from 0.25 to 1.98 A. The variation of these structures with respect to the x-ray structure is 1.89-2.21 A for all residues and 1.58-1.68 A for the ring residues alone. The larger value up to 2.21 A results from the difference in the orientation of the residues in the two ,&turns. The violations of the distance constraints imposed by the NOE data are listed in Table VI. It includes the averaged values of the sum of violations of the upper distance constraints imposed by the NOE data (V,,,) , the number of violations greater than 0.2 A ( V n ) ,and the maximum value of the violations (V,,,) . They were tabulated for all structures including the x-ray structure. This table reveals that the x-ray structure deviates more from the experimental constraints than the five different RMD structures. The reason is the orientation of the turns from the solution structure as prescribed by the experimental constraints. Of the 154 distance constraints imposed in the RMD run, a maximum of 23 violations were observed with a maximum value in the range 0.8-1.33 8, in the case of the RMD structures. The greater values of the violation are contributed by constraints involving the side-chain atoms, especially from the tripeptide tail. With regard to the flexibility of the tail region, it is observed that the precise orientation of the tail end region relative to the rigid tocin ring is seen to be varying. This is further confirmed by the following observations. From the analysis of the dihedral



Table V The RMS Differences Between the Five Selected RMD Structures and the X-Ray Structure of Deamino-Oxytocin Computed for the Main-Chain Atoms" For Cyclic (Tripeptide) Residues RMD Structure X-Ray For All Residues X-ray RMD structure I






1.56 (0.28)

1.62 (0.46)

1.68 (0.69)

1.63 (0.67)

1.58 (0.59)

0.29 (0.45)

0.29 (0.68) 0.44 (0.45)

0.40 (0.56) 0.23 (0.47) 0.44 (0.43)

0.37 (0.49) 0.24 (0.37) 0.44 (0.41) 0.23 (0.32)




















a The values in the upper triangle computed for the cyclic (six) residues. The values given in the parentheses are for the tripeptide residues. The values in the lower triangle refer to all residues.

angles among the RMD structures, there is a minimum variation in the dihedral angles for the tocin ring residues among the structures compared to the tripeptide tail, for which a larger variation is noted; this result gives an indication of the flexible state of the tail-end turn. From the Ramachandran 4-q plot drawn for the residues Ile3, Gln4, Pro7, and Leu8 (Figure 4 ) , four separate regions are marked according to the dihedral angles traced by the four residues in the conformational space generated during the RMD run. Because these residues are the main determinants of the two 0-turn structures, we consider only their dihedral angles. According to this figure the regions of conformational space spanned

by the residues Ile3 and Gln4 are compact, whereas those spanned by Pro7 and Leu8 are wider and scattered. This result implies that the conformations traced by the tail residues are widely changing compared to the smaller range of conformations traced by the ring residues. Hence the tripeptide tail is more

150.0 120.0 90.0

60.0 30.0

Y Table VI Violations of NOE Distance Constraints"


-30.0 -60.0



X-ray RMD I I1 I11 IV V a






(J I:.

0.32 0.39 0.50 0.47 0.37

For explanations see the text.

17 23 17 20 17

0.89 0.91 1.33 1.16 1.08

-180.0 -180.0












a) Figure 4. Ramachandran (p, J / ) plot for the dihedral angles spanned by the residues Ile3 ( * * * ) , Gln4 (000), Pro7 ( 00 a), and Leu8 (-) for the different oxytocin conformations attained during the RMD run.


flexible than the ring portion. Carbon-13 spin-lattice relaxation ( T1) experiments led to a similar conclusion." This flexibility is also confirmed from an analysis of the RMSD values listed in Table V. The upper triangle consists of the RMSD values computed with consideration of only the tripeptide residues (listed in parentheses) with respect to each structure. An analysis of these values for the RMD structures indicates that they are relatively greater than for those residues in the tocin ring. This result further confirms that the tail end residues are more flexible than the ring residues.16,'8.20

CONCLUSION The conformation of oxytocin in DMSO has been determined by means of NOE constraints and molecular dynamics simulation. The ensemble of structures obtained from the RMD method represents a solution structure of oxytocin compatible with all observed experimental data. The obtained conformation of oxytocin is similar to that in the crystal structure of deamino-oxytocin. Our results confirm the earlier studies. The turn consisting of Tyr2 to Asn5 in the 20-memberedtocin ring is stabilized by the disulfide bridge and the hydrogen bond between the carbonyl oxygen of Tyr2 and amide proton of Asn5. Similarly, another turn consisting of residues Cys6 to Gly9 is also stabilized by a hydrogen bond between the carbonyl oxygen of Cys6 and the amide proton of Gly9. Although the solution conformations of oxytocin are comparable to that of deamino-oxytocin, the former possesses twist with respect to the orientation of both turns. This result is confirmed by the RMSD values and the dihedral angles. Furthermore, the solution conformations of oxytocin indicate that the turn in the tail region fluctuates more than the tocin ring. We thank Dr. D. Hare for providing FTNMR software, the referees for their comments and suggestions, and the National Science Council of The Republic of China for a research grant (NSC 81-0208-M-007-62).

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Received February 19, 1992 Accepted April 29, 1992

Conformational properties of oxytocin in dimethyl sulfoxide solution: NMR and restrained molecular dynamics studies.

The conformation of oxytocin, the neurohypophyseal nonapeptide hormone, in solution in deuterated dimethyl sulfoxide has been determined by 1H-nmr. Th...
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