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 <Glu-His-Pro-NH2. This the puckering of the py'rrolidine ring (40-56). The results hormone controls the secretion of thyrotropic hormone and show that in TRF and model peptides the ring is preferen- prolactin from the anterior pituitary gland (1-4). Many in- tially fixed in "endo" (CG syn with respect to C = 0), while vestigations involving synthesis and bioassay of TRF ana- in free proline it seems to be characterized by an averaged logues have been completed in order to determine the struc- planar conformation. tural requirements for activity (5-9). Tritiated TRF has been obtained to allow the study of the cell's binding sites MATERIALS AND METHODS for the hormone (10, 11). Conformational studies, both theo- retical and experimental, are also numerous. The choices of Synthesis. 85% '3C-Enriched Proline. 13C uniformly conformation permitted by this short peptide molecule, labeled proline has been prepared by biosynthesis from which contains a proline residue known to introduce signifi- algae Spirulina maxima (25). cant steric hindrance, are reflected in the various models L-['3C]Proline Amide. l-Ethoxycarbonyl-2-ethoxy-1,2- proposed (12-24). dihydroquinoline (400 mg) and 2.5 M ammonia in chloro- In our previous TRF model, deduced from 1H nuclear form were added to the benzyloxycarbonyl-L-proline (330 magnetic resonance (NMR) studies in hexadeuteriodimethyl mg; prepared from 200 mg of 85% '3C-enriched L-proline) sulfoxide, we showed the angle His to be -150' and stated in chloroform (4 ml) at 0°C. After the mixture was left for 1 that the CO-NH2 terminal could be involved in a hydrogen hr at 0°C and overnight at room temperature the solvent bond with the histidine carbonyl group, leading to a seven- was evaporated, and the residue triturated with ether to membered ring (15). Such a conformation has also been pro- yield 240 mg (73%) of the benzyloxycarbonyl-L-proline posed from energy calculations by Burgess et al. (14). 'SC amide, mp 90-91°C (lit. mp 94°C). This derivative was NMR studies showed that the His-Pro peptide bond adopts treated with a 35% solution of hydrogen bromide in acetic both cis and trans configurations with the trans isomer acid (3 ml) at room temperature for 30 min. The solution being preponderant under all conditions (20-24). 'SC spin was evaporated to dryness; the residue was triturated with lattice relaxation studies demonstrated that there is little ether and dissolved in 50% methanol (5 ml). This solution change in the relative mobilities of the individual residues was passed through a column of Dowex 50 (H+ cycle, 3 ml). upon titration of the imidazole ring of histidine (23). The resin was washed with 50% methanol, and the product Here we report an example of a '3C NMR study dealing eluted with 3% ammonia in 50% methanol. After evapora- tion, the residue was dried azeotropically with benzene to give 94 mg (62% calculated on benzyloxycarbonyl-L-pro- Abbreviations: NMR, nuclear magnetic resonance; TRF, thyrotro- line) of 85% '3C-enriched L-proline amide, mp 94-97°C. pin releasing factor, L-pyroglutamyl-L-histidyl-proline amide; Pro for the 85% '3C-enriched proline. Literature gives mp 99°C (57) and 102-104°C (58) § To whom correspondence should be addressed-Present address: nonlabeled compound. Service de Biochimie, Centre d'Etudes Nucleaires de Saclay, B.P. L-Pyroglutamyl-L-histidyl-L-['5C]proline Amide. The no. 2, 91190-Gif-sur-Yvette, France. coupling was carried out according to Gillesen et al. (59). 4948 Downloaded by guest on September 25, 2021 Biophysics: Haar et al. Proc. Nat. Acad. Sci. USA 72 (1975) 4949 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.