Conformational Studies of Oxytocin, Lysine Vasopressin, Arginine ...

Proc. Nat. Acad. Sci. USA Vol. 70, No. 7, pp. 2086-2090, July 1973

Conformational Studies of Oxytocin, Lysine Vasopressin, Arginine Vasopressin, and Arginine Vasotocin by Carbon-13 Nuclear Magnetic Resonance Spectroscopy (peptide conformation/neurohypophyseal hormone analogs)

RODERICH WALTER*t, K. U. M. PRASAD*, ROXANNE DESLAURIERST, AND IAN C. P. SMITHt *

Department of Physiology, Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029;

t Medical Research Center, Brookhaven National Laboratory, Upton, N.Y. 11973; and t Division of Biological Sciences,

National Research Council of Canada, Ottawa, Canada K1A OR6

Communicated by Maurice Goldhaber, April 11, 1973 Oxytocin, arginine vasopressin, lysine ABSTRACT vasopressin, arginine vasotocin, as well as their cyclic and acyclic analogs, were studied by carbon-13 nuclear magnetic resonance spectroscopy in deuterium oxide and deuterated dimethylsulfoxide. Fourier-transformed spectra were obtained at 25.16 MHz. The resonances of all carbon atoms have been assigned in both solvent systems; this includes tentative assignments of the carbonyl carbons. The spectra of arginine vasopressin and lysine vasopressin are essentially identical when compared in D20 or dimethylsulfoxide, but they differ from those of oxytocin. The spectrum of arginine vasotocin in D20 is intermediate between those of oxytocin and the vasopressins. These spectral differences are not only due to variations in constituent amino acids but are also a reflection of conformational differences of oxytocin, arginine vasotocin, and the vasopressins. All hormones are sensitive to changes in hydrogen ion concentration in both solvents; this was not observed with deamino analogs, which lack the terminal amino group.

Conformational analysis of data from proton nuclear magnetic resonance ('H NMR) spectroscopy has contributed significantly to elucidation of the preferred solution conformations of oxytocin (1, 2) and lysine vasopressin ([Lys8]VP) (3-7). Nevertheless, the conformational analysis involves several approximations: (1) the relationship between the dihedral angle 0 (NH-CaH) and the measured three-bond coupling constant 3JHNCH (8, 9)-a spectral parameter most useful when considered in combination with potential energy maps (10, 11); (2) temperature (12) and solvent (13, 14) dependences of chemical shifts and proton exchange rates (15-17) of peptide hydrogens as related to their solvent-exposed or solvent-shielded state. It is therefore desirable to investigate proposed conformations of peptides by additional spectroscopic techniques. Carbon-13 NMR ('IC NMR) resonances occur over a wide range of chemical shifts, which endows the method with extraordinary structural discrimination (18, 19). '3C chemical shifts (20), spin-lattice relaxation times of individual '3C species (21-23), and couplings between '3C-'H (24-26) and '3C-'3C (27-29) are potentially useful for conformational analysis of peptides. Chemical shifts of 13C resonances allow the detection in peptides of cis and trans isomerism about the amide bonds of proline residues (30-34) and tautomerism in

histidine residues (35); it has also been shown that the helixcoil transition of a polypeptide is accompanied by changes in chemical shifts (36-38). In addition, on formation of a hydrogen bond, downfield shifts of 0.3-0.7 ppm occur for carbonyl '3C resonances (31). Investigation of neurohypophyseal hormones by '3C NMR spectroscopy promises, therefore, to yield valuable information in extending past conformational studies of these peptides by thin-film dialysis (39), chromatographic methods (40), circular dichroism (41), minimization energy calculation (42), and proton NMR (1-7, 43-47). In this paper, we report on the '3C assignment of all a-carbons and side-chain carbons of oxytocin, [Arg8]oxytocin, [Arg']VP, and [Lys8]VP. In addition, we give tentative assignments for the carbonyl resonances of these hormones. Some aspects of solvent effects and pH changes have also been investigated. Similar studies have been performed with a series of neurohypophyseal hormone analogs (unpublished data). The present work was greatly aided by previous '3C assignment of oxytocin and its C-terminal acyclic intermediates in dimethyl sulfoxide [U-3H]Me2SO (31, 48, 49) and D20 (31,48). Our data in water are compared with the tentative assignment of oxytocin and [Lys' ]VP in water by Lyerla and Freedman (50). MATERIALS AND METHODS

Besides the hormones oxytocin, [Arg']oxytocin, [Arg8]VP, and [Lys' ]VP, the analogs deamino lysine vasopressin ([Q3SPP', Lys8]VP), [Ala2]-, [gSPp',Ala2]-, [Orn4]-, [Val4]- and [Val5] oxytocin, and the protected and unprotected acyclic, Cterminal intermediates of oxytocin used previously (31, 48) were studied. Also the nonapeptide intermediates of oxytocin, Z-Cys(Bzl)-Tyr-Ile-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2, and of [Arg8 ]VP, Tos-Cys(Bzl)-Tyr(Bzl)-Phe-Gln-Asn-Cys(Bzl)Pro-Arg(Tos)-Gly-NH2, were examined. Spectra were recorded in 12-mm tubes at 370 on a Varian XL-100-15 spectrometer operating at 25.16 MHz in the pulsed Fourier transform mode with complete proton decoupling. The computers used in these studies (Varian 620-i and Varian 620-L) allowed acquisition of 4096 and 8192 output data points, respectively, in the transformed spectra. Chemical shifts are reported in parts per million (ppm) downfield from external tetramethylsilane. pH values were measured after the experiment (pD = pH + 0.4) (51).

Abbreviations follow the rules of the IUPAC-IUB Commission Biochemical Nomenclature in Biochem. J. 126, 773-780 (1972). All optically active amino acids are of the L-configuration. Me2SO, dimethylsulfoxide; [Arg8]VP, arginine vasopressin; [Lys8] VP, lysine vasopressin; [,3SPp',Lys8] VP, deaminolysine vasopressin ([1-#-mercaptopropionic acid, 8-lysine]vasopressin); [Arg8]oxytocin, arginine vasotocin.

RESULTS AND DISCUSSION The assignment of the '3C resonances of protected oxytocin intermediates in [U-3H]Me2SO was the basis for our initial assignment of the '3C resonances of oxytocin in the same

on

solvent (31, 48). Upon cyclization of the acyclic nonapeptide 2086

Proc. Nat. Acad. Sci. USA 70 (1973)

Carbon-13 NMR of Neurohypophyseal Hormones

Comparison of the spectra of oxytocin with those of the vasopressins shows the carbonyl carbon resonances of all hormones at the extreme low-field region of the spectra, while the 160-100 ppm region exhibits the aromatic carbon resonances. Further upfield are the CH, CH2, and CH3 carbon resonances in this order. The chemical shifts of specific carbon resonances of oxytocin, [Arg]VP, [Lys']VP, and [3SPP , Lys']VP are given in Fig. 1. It can be seen that the portions of the resonances including the Ca of several aminoacid residues common to all three hormones, such as Gln, Pro, Tyr, and Gly, are identical. There are some unexplained differences among the hormones in the resonance positions of C, of Asn and Gln. The spectra of [Arge]VP and [Lys8]VP are remarkably similar. The replacement of the primary amino group of Cys-1 in [Lys8]VP by hydrogen brings abQut an upfield shift of the Ca and C,3 of the residue in position 1. A similar observation has been made by comparison of oxytocin with its deamino analog in Me2SO-d6 (49). The resonances of Ca and C, of Cys-6 are also affected as a result of the deletion of the amino group. The assignments of the carbonyl carbons of the cyclic peptides in Fig. 1 should be considered preliminary.

intermediate to hormone we noted significant changes in the chemical shifts of the Ca and carbonyl carbon resonances of the constituent amino acids of the 20-membered ring of oxytocin as well as of side-chain carbons of the cysteine residues. This observation is reminiscent of findings encountered in the assignment of proton resonances of oxytocin and [Lys']VP (7, 43). The changes that occurred upon cyclization were more pronounced in oxytocin than in [Arg8]VP, which may be associated with the greater conformational freedom of [Arge]VP relative to oxytocin in Me2SO-d6 (4, 7). In making the assignments it was assumed that the order of 'IC resonances of the hormones was the same as in the acyclic nonapeptide for all residues except cysteine. Taking into account the difficulty of following the resonances in the transition from acyclic to cyclic peptide, we refined the assignments of oxytocin by comparing its "IC spectrum with that of the analogs mentioned above. Some of our tentative assignments for 'IC resonances of oxytocin in Me2SO-d6 (48) differ from those of Brewster et al. (49); the variances may be associated with differences in hydrogen-ion content of the samples. We have found that the chemical shifts of certain resonances are strongly dependent upon pH.

Pro.

lie.

Laus GinR

Tyra

Cr1-A.

Pic r

AInR

An a

Pro S

ZIID

UDo

Gly.

Cr,

Twr

Crs-lI

ID

Cys-6D

Lou

ProD

ulerY

GIM

La LeuS

a

1 11

1

iI

1II 1 l

PROTECTED OXYTOCIN NONAPEPTIDE

I

I 1-1 11-

2087

I OXYTOCIN

Tyr

ArgI

II

'

PhgAAin

Cys-6$I 0

c

IArO 1I1,Ag/

PROTECTED

wArg]VP

1111I I4 I i ,'/I

I

/z

I

I~

I

IGln

*

I I

!'11

NONAPEPTIDE

AreS1LyDsr1I

I/

L'Ary

ILP

Tyr/ Ows-l g Lrz Pei 8 1 'I

tI~

I/i \jjcrs. ?

Tyr

(ArgJ VP

'Ii [Lys$] VP

'i

/

I 1111I"

(DSP, Lys'] VP

50

60

I0

20

30

40

Asn

nl

Gly Tyr C4

Tyr C3

Tyr C,

Tyr C2

PROTECTED

OXYTOCI N NONAPEPTI DE

/ I

OXYTOCI N

\ Ph.C2

II

Ph. C5

Ph* C,

I

I

\0h.C4

PROTECTED

CAro] VP NONAPEPTIDE

I

II

Arg

\I

[ArgJ] VP

i

1,

I

I

I

Ii

tLys8] VP

1

11

:1, .

170

160

(DS P. LYs"] VP

11I 140

130

120

110

FIG. 1. Stick diagram of the carbon resonances of oxytocin, [Arg8JVP, [Lys8J- and [,3SPp1,Lys8]VP, and related nonapeptides in [U-'H] Me2SO. Cys-1 and Cys-6 resonances not indicated are believed to be obscured by solvent peaks (center at 41.1 ppm). Chemical shifts of certain resonances depend upon the pH of the aqueous solution from which the hormone was obtained.

2088


Chemistry: Walter et al.

Next we investigated neurohypophyseal hormones in aqueous media. The following approach was used in making the complete assignments of carbons (see Table 1) that includes carbonyl carbons: first, spectra in [U-3H]Me2SO of Nprotected acyclic oxytocin intermediates were compared with either the free base or the hydrobromide of the corresponding peptide. Second, the [U-3H]Me2SO spectra of the same samples of deprotected peptides were matched with their spectra in water. Third, the spectrum of the octapeptide, H-Tyr-Ile-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 (48), was compared with the spectra in water of oxytocin (pH 6.6), [Ala2]oxytocin (pH 7.0), [fSPp1, Ala2]oxytocin (pH 6.5), [Orn4]oxytocin (pH 6.5), and [Val4]oxytocin (pH 6.0). The resonance positions of the spectra of oxytocin taken in [U-3H] Me2SO and D20 are verysimilar, allowing for solvent effects on resonances of certain residues. Our assignments for oxytocin in water (Fig. 2) differ in certain respects from those of Lyerla and Freedman (50), i.e., in the assignment of the Ca resonances of Cys-6, Gln, and C,6 resonances of Cys-1 and Lys.

60

40

20

ARGININE VASOTOCIN Cys -Tyr-Ile-Gin - Asn-Cys-Pro-Arg-Gly-NH2 D20 pD 3.5

60

40

20

-~~~~~~ U LYSINE VASOPRESSIN Cys-Ty -Phe - GIl - Asn- Cys- Pro-Lys - Gy -NH2

DO pD3.9

60 ppm

20 40 from external tetramethylsilane

FIG. 2. 13C spectra (aliphatic region) taken in D20, of: oxytocin, pD 3.0, 50 mg/ml, 25,873 transients; [Arg8] oxytocin, pD 3.5, 38 mg/mnl, 85,634 transients; [Lys8]VP, pD 3.9, 40 mg/ml, 125,496 transients.

The '3C spectra of [Arg8]oxytocin, [Arg8]VP, [Lys8]VP, and [13SP l, Lys8]VP were assigned by comparison with the fully assigned spectrum of oxytocin at the same pH. The spectra of all four hormones, which can be viewed as structural analogs of each other, were then inspected for internal consistency of assignments. This approach was efficient and reliable for the assignment of neurohypophyseal hormones by proton resonance spectroscopy (7, 45). The high-field region of the spectrum of [Arg8]oxytocin is essentially the same as that of oxytocin, taking into account the-presence of an Arg instead of a Leu residue in position 8. Notable, however is the difference of 0.2 ppm between Ca resonances of the Ile residue in each hormone at the same pH. The Ca resonances of Asn, Gln, and Cys-1 differed by 0.1 ppm in these hormones, which may reflect a loosening of the 20-membered ring conformation of [Arg8]oxytocin caused by charge repulsion of formally-charged terminal amino groups and the guanidino moiety of arginine. The spectra of [Arg8]oxytocin and [Arg8]VP are also similar after adjustments have been made for the replacement of an Ile by a Phe residue. The Ca and Cp resonances of Asn in both hormones differed by 0.3 and 0.4 ppm, respectively, and the Ca of Gln differed by 0.2 ppm. As expected, the spectra of [Arg8]VP and [Lys8]VP are for all practical purposes identical. Having completed the assignments of all carbons of the hormones and discussed spectral aspects and conformational implications, it was of interest to investigate the effect of progressive changes of hydrogen-ion concentration. As the pH was increased stepwise from 2.6 to 8.3, two major effects were observed with all hormones. As illustrated with oxytocin, the largest chemical shift changes occurred in the pH range 5-7.5, which is in the range of the apparent pK of its amino group (pK = 6.3) (52). The resonances of carbons of cystine show the most significant changes, e.g., the C, resonance of the half-cystine in position 1 shifts downfield by 3 ppm, and the Ca shifts downfield 1.7 ppm. The carbonyl carbon also undergoes a significant downfield shift (5 ppm). Such a pHdependent shift of "3C resonances, which is smaller for the carbon bearing the ionizable amino group than for carbons in ,3-position to that group, was studied in detail by Floh6 et al. (53). The chemical-shift changes have been associated with alterations in the electron density on the carbons and, on the basis of semiempirical molecular orbital calculation by Del Re et al. (54), such a polarization would be most strongly felt at (3-carbons. The polarization effects can occur through the bonds of a saturated system as an inductive effect and through space as an electric effect-the polarization through space being the more far-reaching effect (55). Deprotonation of the amino group also affects the chemical shift of the half-cystine residue in position 6. The (3-carbon shifts downfield by 0.8 ppm and the a carbon shifts downfield 0.5 ppm. The second noticeable effect involves the Ca of Ile, which shifts upfield 0.7 ppm. Thus, the deprotonation of the amino group is felt seven bonds away. Changes up to 0.5 ppm in the (3-carbon resonances of the Tyr, Ile, Phe, Gln, and Asn residues in the 20-membered ring may also be a consequence of conformational changes in oxytocin (and [Lys8]VP) during the pH titration. In sharp contrast, resonances of residues present in the acyclic portion of the hormones are not affected during a change of pH from 2.6 to 8.3. Moreover, [USPpl,Lys8]VP and [,OSP.1,Ala2]oxytocin, which lack the terminal amino group, fail to show any dependence of chemical shifts of the cystine residue in the pH


Carbon-13 NMR of Neurohypophyseal Hormones

2089

TABLE 1. Assignments of 'IC resonances of oxytocin, [Arg8]oxytocin, [Arg8]VP, [Lys8]VP, [j3SP,',Ala2]oxytocin, and [,#SP,,,Lys8]VP in D20

O X Y T 0 C I

PD Cly

1CH CEO

Leu