Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, pp. 4948-4952, December 1975 Biophysics 13C-nuclear magnetic resonance study of [85% 13C-enriched proline]thyrotropin releasing factor: 13C-13C vicinal coupling constants and conformation of the proline residue (hypothalamic hormone/13C labeling/pH effects/angular dependence/pyrrolidine ring puckering) WOLFGANG HAAR*, SERGE FERMANDJIAN*§, JAROSLAV VICARt, KAREL BLAHAf, AND PIERRE FROMAGEOT* * Service de Biochimie, Centre d'Etudes Nucleaires de Saclay, B.P. no. 2, 91190-GIF-sur-Yvette, France; t Institute of Organic Chemistry and Biochemistry, Flemingoro Namesti 2, Prague, Czechoslovakia; and * Institute for Medical Chemistry, Palacky University, Olomouc, Czechoslovakia Communicated by Irvtine H. Page, September 22, 1975

ABSTRACT To understand fully interactions between with a natural peptide containing a single '3C-enriched peptides and cellular receptors, peptide side chain conforma- amino acid. In previous papers, we have demonstrated that tion must be defined. In many cases the complexity of proton 85% '3C-enrichment of amino acids offers several advan- nuclear magnetic resonance (NMR) prevents this but the tages (25-27) when compared to unenriched samples. Not present work demonstrates this problem can be solved by also those of using 13C enrichment. Selective '3C enrichment of a natural only are problems of concentration solved, but peptide hormone has been achieved by preparing [85% '3C signal assignment if only one amino acid is selectively la- enriched prolinelthyrotropin releasing factor which was ex- beled in the peptide. Furthermore '3C-'3C coupling con- amined by '3C NMR spectroscopy at various pH values. Be- stants, undetectable with unenriched samples, are easily cause of the '3C enrichment, one-bonded and three-bonded measured (28-89). (vicinal) "3C-'3C coupling constants have been determined. Because of the general conformational interest of the pro- The latter vary from 0 to 5 Hz and show bond angle depen- dence. These data indicate that in this hormone the pyrrol- line residue, the study of [85% '3C-enriched proline]TRF idine ring is not free but fixed in the Cl-endo puckered con- has been undertaken. Both the 13C chemical shifts and the formation. It has also been possible to assign chemical shift 13C-13C coupling constants in TRF have been examined as a values for a second order 13C NMR spectrum. function of pH in Gly-Pro compared to Gly-Pro-Gly. (13C- enriched Pro is designated by Pro.) The 3Jisic_v have been Thyrotropin releasing factor (TRF) is a hypothalamic, tri- interpreted in terms of angular dependence for analysis of peptide hormone with sequence

CcaCL& 50 Hz Jcc,

(a)

co

J (tran.) J (Cis) Cy (b) I 200Hz

(C)

FIG. 1. (a) Ca, C6, Ca, and C, 13C-multiplets of the proline residue in [13C-Pro]TRF obtained with a spectral width of 1000 Hz. Bridges indicate the vicinal coupling constants. (b) 13C spectrum of the proline residue in [13C-Pro]TRF. Bridges indicate the one-bond coupling constants. (c) Calculated 13C spectrum of the trans-proline residue in [13C-Pro]TRF. Signals due to the enrichment deficit have been ne- glected.

Starting from 143 mg of L-pyroghlitamyl-L-histidine hydra- eral solvent systems and could not be distinguished from the zide and 62 mg of L-[13C]proline amide, 63 mg (35%) of the nonlabeled specimen. tripeptide were obtained. Amino-acid analysis: Glu 1.08, His 13C NMR Measurements. The enriched compounds were 1.0, Pro 0.91. The product was determined to be pure by dissolved in 2H20 in the concentration range of 0.1-0.2 M. thin-layer chromatography and paper electrophoresis in sev- The pH of the samples was adjusted with concentrated solu- Downloaded by guest on September 25, 2021 4950 Biophysics: Haar et al. Proc. Nat. Acad. Sci. USA 72 (1975) Table 1. Chemical shifts* of the 13C resonances of the proline residue in "13C-Pro] TRF in 2H20 solution CO C0CCo C.) C6 trans cis L5Tct trans cis A5TCt trans cis .AbTCt trans cis AbTCt trans cis A5TCt pH= 1.6 177.59 177.07 0.52 61.30 61.45 --0.15 30.48 32.73 -2.25 25.44 22.59 2.85 48.86 47.98 0.88 pH= 9 177.65 177.03 0.62 61.38 61.17 0.21 30.32 32.29 -1.97 25.37 22.66 2.71 48.80 47.98 0.82 A\61.6-9f -0.06 0.04 - -0.08 0.28 - 0.16 0.44 - 0.07 -0.07 0.06 0 * Chemical shifts 6 in ppm from tetramethylsilane. 1 6TC = btrans - 6cis. t 61.6-9 = b(pH = 1.6) - 6(pH = 9).

tions of 2HCl and NaO2H. None of the pH measurements the '3C natural abundance compound appear at this posi- was corrected for deuterium isotope effects. '3C NMR spec- tion. For the second-order spectrum (C13 - C7), theoretical tra were obtained on a Varian XL 100 12 WG and a CFT 20 spectra taking into account only the '3C-'3C-'3C combina- spectrometer in the Fourier Transform Mode with complete tions have been computed to determine the exact chemical proton decoupling, using 16 K computers. The sample-under shifts (Fig. ic). Table 1 presents the values of the '3C chemi- study was placed in a tube of 10 mm outer diameter; diox- cal shifts of the proline residue in the trans and cis forms, ane was used as internal reference. Chemical shifts are re- obtained at two pH values corresponding to two states of ported downfield from tetramethylsilane. For all measure- protonation of the imidazole ring: imid+ (pH 1.6) and imid ments, 2H20 used as solvent provided the deuterium lock (pH 9) (23). Owing to the 13C enrichment, the Co cis isomer signal. Spectra were recorded at about 30'C, and theoretical signals have also been easily detected. The pH effect on the spectra have been calculated by the LAOCN program. chemical shifts of the '3C resonances is weak in each form although the cis Ca and C, are the most affected. It is inter- RESULTS AND DISCUSSION esting to note that while the Ca carbon does not appear to be Interpretation of [13C-Labeled ProlineTRF Spectrum. perturbed, the results obtained by 'H NMR show changes in Fig. la and b shows the spectra of the 85% 13C-enriched the b-proton chemical shifts (13). proline residue of TRF obtained in D20 solution (pH = 7) 13C-13C Coupling Constants. (a) One-Bonded Coupling with spectral widths of 1000 and 4000 Hz, respectively. The Constants. Two types of one-bond coupling constants are in - spectra are complicated by a large number of signals arising found amino acids: the sp2 sp3 (JcocG 50-60 Hz) from two important phenomena. First, the 85% '3C-enrich- and the Sp3 - SpS (Jccs, Jc,-c7, Jcc,-ca 30-35 Hz). All ment ratio causes one, two, and three bond range '3C-'3C these couplings can be measured as indicated by the bridges coupling combinations because of the 13C or '2C nuclei of on the spectrum (Fig. lb) and checked with the theoretical adjacent, geminal, or vicinal sites in the carbon chain. The spectrum (Fig. ic). The one-bond coupling constants ob- intensities of the lines are proportional to the probability of tained from the '3C-enriched proline residue in TRF are each of these combinations (25-27). The second complicat- presented in Table 2. These are about the same as those de- ing phenomenon is the cis-trans isomerism of the pyrrol- termined for proline and the proline residue in Gly-Pro and idine ring. As in many other peptides containing the proline Gly-Pro-Gly (26). In this latter compound and in TRF they It in residue, two conformers can be seen as a consequence of the are only weakly dependent on pH. seems that the hor- small difference of stability existing between the cis and mone the ionization state of the histidine imidazole ring does trans forms generated by the rotation around the imide bond not significantly affect the couplings occurring in the pro- (60-67). The ratio of the signals indicates that about 20% line residue. The difference of about 1.4 Hz existing be- Cb cis of the cis isomer is present in TRF and in Gly-Pro-Gly, tween Jco-c0 and Jco-c0 trans (cis smaller than trans) to a is in whereas in Gly-Pro this percentage of cis is found only at appears be significant, for similar difference found acidic pH (20-24). Gly-Pro-Gly. This means that the phenomenon is not due to special effects of the imidazole ring in TRF or of the NH2- Another difficulty arises from the fact that the - G C,1 in In spectrum appears as second order: n 0.3. Even for the and COOH-terminal group charges Gly-Pro-Gly. both J/Av compounds the difference observed between and C,- C systems, where J/Av - 0.03, there is some tenden- J* Jtran, cy to deviate from the first order spectrum, for the lines of might be generated by the orientations of the carbonyl Cb multiplet do not correspond to strict binomial intensities. groups of the histidine and the proline residues, respectively, Chemical Shifts. The chemical shift of the 13C. reso- nance of the proline residues in TRF (first-order spectrum) Table 3. '3C-13C vicinal coupling constants (in Hz) in is given by the center of the multiplets arising from the proline and the proline residue in peptides* number of possible coupling combinations (Fig. la, b). The noncoupled 13C signal and also the single line resonance of 3JC.C-S 02 (X2) 3JCOc.e 01

Pro t 1.5 1250 Table 2. One-bond 13C- 13C coupling constants (in Hz) Gly-Pro 4.4 ±20° >0.5 90-1000 of the proline residue of TRF Gly-Pro-Gly 4.3 ± 22° >0.5 90-1000 [13C-Pro]TRF 3.9 +300 >0.5 90-1000 lc- < Jc

C ICO \c N..Cw N - -C N \\__r-CA1p ,,, a

H C'v

C7

EXO PLANE ENDO FIG. 2 Preferential conformations of the pyrrolidine ring and the corresponding approximative values of 3Jco c. and 01 (Co, Ca, C15, C').

which are arranged cis and trans to each other in the two 300, and X4 --150 (40-56). On the other hand, free pro- molecular forms. Moreover, it is also possible that, due to the line, which is known to exist in equilibrium of rapidly inter- difference in steric hindrance or in interactions of the converting puckered forms, gives a value of 1.5 Hz for COOH-terminal amide group, the *Pro value differs in the 3Jco-c coupling. This value corresponds to the mean angle of cis and the trans isomers of the peptides. 01 of 1200 determined from the curve for butanoic acid (37), (b) Three-Bonded (Vicinal) Coupling Constants. The which seems to be more suitable for this coupling. Applying two-bonded (geminal) couplings have been shown to be the curve for 2-butanol (37), this coupling constant gives a small in saturated amino acids (27). They have not been ob- value for 91 lying between 1200 and 135", very close to that served in the proline residue between carbons where only calculated from the former curve. Thus our results indicate geminal couplings are possible. Therefore, it seems justified that the proline residue in TRF is preferentially in the endo to consider the couplings found in the pyrrolidine ring to be conformation in contrast to proline itself. However, a small vicinal, although they might be either geminal or vicinal. contribution of the exo-conformer, which might explain the Theoretically, five vicinal couplings should be measured 13C relaxation time data (21-23), cannot be totally excluded in the proline spectra (3Jco-c,, 3JGO-_C,3JCG-CY, 3JCaGCS and as long as more accurate relationships between 3Jc-c and di- 3Jc15-cb). However, not all of them could be determined, for hedral angles are lacking. their observation was prevented either by the large number According to the calculations of Pullman and Pullman of splittings and the interference from other spectral lines (47) on the proline residue in peptides, such a puckered ring (without 13C homonuclear decoupling this problem will be is the best conformation leading to formation of a seven- difficult to solve) or because some values lie below the in- membered hydrogen bonded ring between the COOH-ter- trinsic resolution (0.5 Hz). The 3Jc-c determined in the pro- minal amide NH and the oxygen atom of the preceding line residue and in proline are indicated in Fig. la. The peptide bond (68, 69). This would support the TRF model values presented in Table 3 are in good agreement with previously reported (hydrogen bonding involving the histi- those generally admitted for the vicinal coupling constants dine C = 0 group and the NH2 of the COOH-terminal (27-37). amide group) based both on 1H NMR and calculation stud- Angular Dependence of Vicinal Coupling Constants. ies in which the pyrrolidine ring conformation had not been Barfield et al. (37) have proposed a plot of the calculated specified (14, 15). vicinal '3CG-3C coupling constants in 2-butanol and butanoic The point to emphasize is that the experimental results acid as a function of the dihedral angles. Using our coupling presented here are in good agreement with the calculation data with these curves, we determined the two dihedral an- and x-ray data for the conformational behavior of the pro- gles 0' (CO, Ca, CG, G) and 92 (Ca, C,, CG., G) defining the line residue within a polypeptide chain. It also appears that, conformation of the pyrrolidine ring (Table 3). The 02 (X2) whereas it is difficult to elucidate the conformation of the value (L 300) given by 3Jca,-c reflects the large distortion of proline residue by 1H NMR (considering the great number the ring planarity in the peptide but does not permit distinc- of protons and the ambiguities of the interpretation of vici- tion between fixed exo or endo conformations and rapid nal couplings), 13C NMR using '3C-enriched samples is a equilibrium between the two. This problem could be solved powerful instrument. by the 91 value (given by 3Jc0-c,) which is also related to the degree of puckering of the pyrrolidine ring. The value close 1. Schally, A. V., Arimura, A., Bowers, C. Y., Kastin, A. J., Sawa- to zero found for this coupling in TRF corresponds to a dihe- no, S. & Redding, T. W. (1968) Recent Progr. Horm. Res. 24, dral angle of about 900 using the curves for 2-butanol and 497-588. butanoic acid of Barfield et al. (37). Looking at a molecular 2. Folkers, K., Enzmann, F., Boler, J., Bowers, C. Y. & Schally, model, both the angles 01 = 90° and 92 (x2) -30°C are A. V. (1969) Biochem. Biophys. Res. Commun. 37,123-126. compatible with a C y-endo puckered pyrrolidine ring (Fig. 3. Burgus, R., Dunn, T. F., Desiderio, D. M. & Guillemin, R. 2). This conformation found in solution and in crystals of (1969) C. R. Hebd. Seances Acad. Sci. Ser. D. 269, 1870-1873. peptides containing proline, and calculated by several au- 4. Gourdji, D., Kerdelhue, B. & Tixier-Vidal, A. (1972) C. R. thors is defined by - -750, X' 30°, x2 -30" Hebd. Seances Acad. Sci. Ser. D. 274, 437-440. Downloaded by guest on September 25, 2021 4952 Biophysics: Haar et al. Proc. Nat. Acad. Sci. USA 72 (1975)

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