Proc. Nati. Acad. Sci. USA Vol. 77, No. 1, pp. 82-86, January 1980 Biochemistry Amino acid side chain conformation in angiotensin II and analogs: Correlated results of circular dichroism and 1H nuclear magnetic resonance (peptide hormones/competitive inhibitor/N-methylation/pH titration) F. PIRIou*, K. LINTNER*, S. FERMANDJIAN*, P. FROMAGEOT*, M. C. KHOSLAt, R. R. SMEBYt, AND F. M. BUMPUSt *Service de Biochimie, D)partement de Biologie, Centre d'Etudes Nucl&aires de Saclay, P.B. No. 2, F-91190 Gif-sur-Yvette, France; and tThe Clinic Center, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44106 Communicated by Irvine H. Page, September 4,1979 ABSTRACT [1-Sarcosine,S-isoleucinelangiotensin II (Sar- [Sarl,11e8]Angiotensin II was shown to be a potent antagonist Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent an- of the pressor action of antiotensin II (Asp-Arg-Val-Tyr-Ile- tagonist of the pressor action of angiotensin II. With a view to increase half-life in vivo of this peptide, the amino acid residue His-Pro-Phe). However, this and other similar antagonistic at position 4 (tyrosine) or position 5 (isoleucine) was replaced peptides have short half-lives in vivo; the shortness is presum- with the corresponding N-methylated residue. This change ably due to rapid degradation by peptidases (for detailed re- drastically reduced the antagonistic properties of this analog. views, see refs. 1 and 2). With a view to render these peptides The present work was therefore undertaken to investigate the more resistant to enzymatic degradation, the residue at position effect of N-methylation on overall conformation of these pep- tides and to determine the conformational requirements for 4 (tyrosine) or position 5 (isoleucine) was replaced with the maximum agonistic or antagonistic pro rties. Conformation corresponding N-methylated amino acid. The analogs thus studies were carried out by circular dichroism and proton nu- synthesized, [Sar',MeTyr4,I1e81- and [Sar1,MeIle5,Ile8jangio- clear magnetic resonance spectroscopy in aqueous solution as tensin II showed drastically reduced antagonistic properties (3, a function of pH. The results indicated that: (i) angiotensin II to N-methylation and [1-sarcosine,8-isoleucinelangiotensin II gave practically 4). It is possible that the reduced activity due identical spectroscopic data; and (ii) N-methylation in either may be a consequence of either modification of backbone position 4 or position 5 resulted in remarkable changes in the conformation or rotational restriction of the side chain, or both. peptide backbone and a severe limitation in rotational freedom It has been shown that N-methylation of a single amino acid of side chains in tyrosine, isoleucine, and histidine residues. residue in a peptide chain restricts the number of conformations However, rotational restriction of the tyrosine side chain was found to be less pronounced in [1-sarcosine,4-N-methyltyro- not only of the N-methylated residues but also of the residue sine,S-isoleucinelangiotensin II than in [1-sarcosine,5-N- preceding it (5, 6). Further, N-methylation may also affect the methylisoleucine,S-isoleucinejlangiotensin IL Thus, these results lowest energy conformation of the involved fragment. There- suggest that: (I) the backbone and side chain structure of a potent fore, the conformations of the above analogs and those of an- angiotensin II antagonist should resemble that of the hormone, angiotensin II, so that it can mimic the hormone in recognizing giotensin II and [Sar',Ile8Jangiotensin II in water solution were and binding with the receptor on the cell membrane; and (ii) examined as a function of pH. Circular dichroism (CD) and greater impact of N-methylation in position 5 on the overall proton NMR spectroscopy were used to obtain information conformation of these peptides points to the controlling influ- about the side-chain conformations, especially with regard to ence of position 5 (isoleucine) in aligning the residues in the those residues that are important for biological activity, namely, central segment (tyrosine-isoleucine-histidine) of angiotensin II and its potent agonist and antagonist analogs in a nearly ex- tyrosine and histidine. tended structure. Any change in this arrangement may lead to reduced biological activity. MATERIALS AND METHODS The synthesis and purification of the peptides have been de- Recognition of a biologically active molecule by its receptor site scribed (3, 4). CD spectra were recorded on a Dichrograph is probably the first event of the hormone-receptor interaction concentrations were process, whereas binding and signal release are subsequent model Mark III (Jobin Yvon). Peptide phenomena. Thus, it is likely that during the approach of a made to about 0.5 mM and were determined by ultraviolet peptide to the cell membrane the local environment may absorbance (E275 = 1350 M-' cm-' at pH 5.8). The previously greatly influence the selection of a "recognizable" conformation described method of pH titration was used without modifica- among numerous other conformations. It is therefore assumed tion (7). Theoretical titration curves were calculated with the that the peptides showing physical properties similar to those help of a computer program "Titrage" kindly made available of the parent hormone, in a given set of experimental conditions by M. Gingold. Results are expressed in molar ellipticity [0I = (e.g., solvent and pH effects, etc.), may survive the conforma- 3300 Ae, in units of ocm2/dmol. tional selection procedure during their approach to the cell 'H NMR spectra were recorded at 250 MHz on a CAMECA membrane. The present work was undertaken to provide evi- TSN 250 instrument in the Fourier transform mode. The dence for this hypothesis, by using angiotensin analogues as peptide concentration in 2H20 was 35 mM. The internal ref- model peptides. erence was sodium 2,2,3,3-tetradeutero-3-trimethylsilylpro- pionate; the lock was on 2H; and the temperature was 220C (except for the ca protons of histidine in [Sarl,Ile8langiotensin The publication costs of this article were defrayed in part by page was to shift the HO2H charge payment. This article must therefore be hereby marked "ad- II, for which 40'C necessary peak). vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: CD, circular dichroism. 82 Downloaded by guest on September 25, 2021 Biochemistry: Piriou et al. Proc. Natl. Acad. Sci. USA 77 (1980) 83 RESULTS Histidine Titration. Arguments based on data from a large CD number of analogs studied (9) have shown that the inversion Marked differences were observed in the CD spectra of the four of the tyrosine signal in angiotensin II (Fig. lB) is due to the peptides dissolved in water at pH 5.8, recorded in the aromatic titration of the close-up histidine side chain. The amplitude of region (320-250 nm). The sign and the intensity of the tyrosine the titratiop curve observed for a given peptide is a function of signal at the initial pH value (5.8) are given in Fig. 1, in which its conformational characteristics especially with respect to the the values for [Sar',MeIle5,Ile8]angiotensin II (+450) and central residues 3 to 7 of angiotensin 11 (8). The curves in Fig. [Sarl,MeTyr4,Ile8langiotensin II (-850) are noteworthy. Signal 1B follow this pattern. Angiotensin II and its potent inhibitor, intensity, which in tyrosine model compounds and previously [Sarl,Ile8]angiotensin II, have curves that are almost superim- studied angiotensin II analogs was found to be in the +300 posable (A[0J = -450), which confirms the titration behavior range, is of limited diagnostic value and indicates at best the of the less-modified inhibitor [Ile8]angiotensin II (8). A marked amount of local asymmetry or hindered rotational freedom of reduction in the side-chain-side-chain influence is observed the chromophore. On the other hand, the comparison of titra- for [Sar',MeTyr4;Ile8]angiotensin II (A[O] = -200). The in- tion curves obtained for different peptides yields precise tensity of the 'Lb band in this peptide hints at hindered rotation structural information (8). This is also true for the peptides of the tyrosine side chain, which could account for an increased presented here, for which we shall discuss the three zones of pH time-averaged distance between the two aromatic side chains shown in Fig. 1. tyrosine and histidine. Again, these factors are possibly tied in Carboxyl Titration. The intensity of the 'Lb signal of tyrosine with as yet unquantifiable changes in backbone conformation. in angiotensin II is sensitive to the titration of one or more The titration curve for [Sarl,MeIle5,le8]angiotensin II, on the carboxyl groups (Fig. 1A) (8). It had been observed that the other hand, is flat in this pH region; the respective orientation slope of ihe titration curve is dependent on the presence or of the tyrosine arnd histidine side chains and the distance be- absence of the f3-carboxyl group in position 1: it is positive in tween them has clearly been upset by the N-methylation and [Aspl]angiotensin II peptides but negative in [Asn']angiotensin the concomitant changes of dihedral angles 4 and i/' along the II, [Me2Glyl]angiotensin II, and des-Aspl-angiotensin II, etc. central stretch of the backbone. (8). Whereas the possibility of conformational changes in the Phenol Titratidn. It is only between pH 9 and pH 12 that we backbone structure during COOH titration or an indirect titrate the chromophore of the tyrosine side-chain proper. The effect via the histidine side chain cannot be denied, the relative change in electronic structure causes the well-documented shift distance and orientation of the carboxyl group toward the ty- of the 'Lb band from 275 nm to 293 nm; in almost all cases so rosine side chain is likely to be the main factor leading to the far published in the literature or observed in our laboratory observed effects.