Proc. Nati. Acad. Sci. USA Vol. 77, No. 1, pp. 82-86, January 1980 side chain conformation in II and analogs: Correlated results of circular dichroism and 1H nuclear magnetic resonance ( 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 () or position 5 () was replaced 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 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. In the event of similar backbone structure, this (scores of the most diverse tyrosine-containing peptides), this distance is primarily determined by the time-averaged orien- 'Lb dichroic band of Tyr-0- was observed to be positive with tations of the tyrosine side chain. In this light, the curves shown ellipticities between 300 and 800 units. The curves for angio- in Fig. 1A are strongly significant. The slope for angiotensin tensin II, .[Sar',1le8]angiotensin II, and [Sar',MeIle5,Ile8]an- II is positive (Aspl), the one for [Sarl,11le8]angiotensin II is giotensin II (Fig. 1C) are therefore quite normal. The surprising negative (a COOH, COOH-terminal). In [Sar1,MeIle5,Ile8]- high intensity negative band found for [Sarl,MeTyr4,Ile8]- angiotensin II the carboxyl titration has an enormous impact angiotensin II must then be seen as a sign of distinctly different on the CD signal: A[OJ = -230 compared to A[6] = -20 side-chain orientation vis a vis its environment. for [Sarl;Ile8]angiotensin II. The tyrosine signal in [Sarl, 'H NMR MeTyr4,Ile8]angiotensin II, on the other hand, is removed from Chemical Shifts. The resonances in the spectrum of angio- any influence of the carboxyl titration. tensin II were assigned on the basis of double resonance ex- periments and pH titration effects; the assignments agree well with data published previously (10). They serve as basis for the signal assignment in the analogs discussed below. Chemical shifts of the resonances in the competitive inhibitor [Sar',1le8]- angiotensin II are quite similar to those found in angiotensin II, with the exception of those for Sarl and Ile8, of course. The N-methylation of residues in positions 4 and 5-e.g., in [Sarl,MeTyr4,le8]angiotensin II and [Sarl, MeIle5,Ile8]angio- tensin II-leads to sharply reduced inhibitory activity; this change also has profound effects on the NMR spectra: The resonances of the a protons of the N-methylated residue and those preceding it in the sequence are considerably shifted to lower fields (0.3 to 0.7 ppm), whereas the a protons of the res- idue succeeding the N-methylated residue undergo shifts of 0 < Ab < 0.3 ppm (Fig. 2). The resonances of further removed residues are not affected compared to [Sarl,Ile8Jangiotensin II, except for the a proton of the COOH-terminal isoleucine such as in [SarlMeIle5,11e8]angiotensin II, for which a 0.39-ppm downfield shift was registered. Whereas the immediate causes for the observed chemical shift variations between these pep- tides may lie in variations of electronic density and local pH modifications of steric hindrance due to the effects of -CH3, other factors FIG. 1. Titration curves. For A and B, ellipticity was measured responsible for the observations will be discussed at 275 nm; for C, 293 nm. A, Angiotensin II; *, [Sarl,l1e8]angiotensin below. II; o, [Sar',MeTyr4, Ile8]angiotensin II; *, [Sar',MeIle5,Ile8Jangio- Coupling Constants. Whereas the spectrum of [Sar',Ile8J- tensin II. angiotensin II at 250 MHz allows the various /-proton signals Downloaded by guest on September 25, 2021 84 Biochemistry: Piriou et al. Proc. Natl. Acad. Sc. USA 77 (1980)

N I 1E '0

1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 7 9 11 pH FIG. 2. Titration curves, chemical shifts (6) against pH. (A-D) Aromatic protons: (A) angiotensin II; (B) [Sar',Ile8]angiotensin II; (C)

[Sar1,MeTyr4,I1e8]angiotensin II; (D) [Sar'MeIle5,lle8]angiotensin II. 0, C2H His6; 0, C4H His6; A, C6H5 aromatic Phe8; *, CH m-Tyr4; 0, CH o-Tyr4. (E-H) Aliphatic protons: (E) angiotension II; (F) [Sarl,Ile8Jangiotensin II; (G) [Sar',MeTyr4,Ile8jangiotensin II; (H) [Sarl,Me- Ile5,Ile8]angiotensin II. *, CaH His6; O, CaH Tyr4; 0, CaH Phe8 or Ile8; *, CaH Pro7; X, CaH Arg2; +, CaH Asp' or Sari; A, CaH ValW; v, CoH

Ile5; *, C5H Pro7; A, C6H Pro7.

(AB parts) to be observed clearly, signal overlapping in an- with the chemical shifts. On the other hand, the NMR param- giotensin II-e.g., in [Sar',MeTyr4,Ile8]angiotensin II and eters of [Sar',MeTyr4,I1e8]angiotensin II are different from those [Sar',MeIle5,Ile8]angiotensin II-precludes exact analysis of of [Sarl,l1e8]angiotensin II principally with respect to the ty- this region. The 1H NMR spectrum of angiotensin II obtained rosine side chain, but also with respect to Ile5 and, more sur- at 360 MHz, however, renders complete analysis of the ABX prisingly when we consider the chemical shift effects discussed patterns possible (unpublished results). For the other two above, the twice-removed His6 residue. The most drastic effects, peptides, coupling constants were measured on the a-proton however, are observed for [Sarl,MeIle5,Ile8jangiotensin II, in signals. Computer calculations (program "ITRCAL," Nicolet which the side-chain orientations of Tyr4, IVe5, and His6 are still more Hz Instrument Corporation, Madison, WI) on the 360 Mhz and strongly perturbed. The coupled constant of 11.3 for 250-MHz spectra show the results obtained from measuring the lie5 translates into a rotamer II fraction of 0.8 (X = 180°) and indicates almost total absence of rotation. For tyrosine and 3JC-H-GH coupling constants on the signals to be valid. a-proton histidine it seems reasonable, too, to interpret the coupling These coupling constants are indicators of side-chain orientation constants (10.0 and 4.8 Hz) in terms of a unique X angle of 0° and are usually used to determine rotamer populations or the or 120°, even though this would correspond to the eclipsed direct value of the Xi dihedral read from a suitable angle conformation. Steric factors originating in the N-methyl group Karplus curve (11). The data observed for angiotensin II and would easily account for this, though. Clearly, the conforma- its analogs are listed in Table 1 together with the respective tional properties of angiotensin II show a higher sensitivity to rotamer fractions. Assignment of rotamers I and II was cori- N-methylation in position 5 than in position 4. firmed by 13C NMR spectroscopy of uniformly '3C-enriched Titration Curves. Proton chemical shifts are sensitive to residues in angiotensin II peptides (13). For angiotensin II, the charge variations in their proximity and thus to pH titrations rotamer distribution varies with the nature of the side chain, of ionizable groups, if they are close because of chemical but not significantly with pH [rotamer I predominated in Aspl, structure or spatial/directional preferred orientation. The pH Tyr4, and Phe8; rotamer II in Val5 and Ile5; rotamers I and II titration curves of proton resonance signals constitute a powerful are preponderant in His6 (Table 1)]. The same values are found means to assign signals to specific residues and to determine for the side chains in [SarlIle8langiotensin II, confirming the spatial proximity between atoms or groups that are structurally conformational homogeneity with angiotensin II already noted distant. Downloaded by guest on September 25, 2021 Biochemistry: Piriou et al. Proc. Natl. Acad. Sci. USA 77 (1980) 85 Table 1. Coupling constants (J) and rotamer (R)* fractions of angiotensin II and analogs [Asp',Ile51- [Sar',Ile8J- [Sar1,MeTyr4,Ile8]- [Sar',MeIle5,Ile8]- Angiotensin II Angiotensin II Angiotensin II Angiotensin II Residue 3JH--HO RI RII 3JH"-Ht RI RII 3JHI-HI RI R11 3JH"-HO RI RII Asp 8.0 0.49 5.0 0.21 Arg 7.0 0.40 7.0 0.40 7.0 0.40 7.0 0.40 7.0 0.40 7.0 0.40 7.0 0.40 7.0 0.40 Val 8.2 0.51 8.5 0.53 8.1 0.50 8.0 0.49 Tyr 8.5 0.53 8.1 0.50 9.0 0.58 10.Ot 0.67 6.5 0.36 6.8 0.38 6.0 0.31 4.6 0.20 Ile 8.0 0.49 8.6 0.54 8.0 0.49 11.3 0.80 His 6.0 0.31 6.6 0.36 7.2 0.42 10.Ot 0.67 5.5 0.27 5.6 0.27 7.2 0.42 4.8 0.20 Pro 8.5 8.8 8.6 8.8 5.0 5.8 5.5 4.7 Ile or 8.0 0.49 6.0 0.31 6.0 0.31 6.5 0.35 Phe 6.0 0.31 * X =-60° (RI), 1800 (R1I), 600 (RI,,) (12). tX = 0 or 1200 is an alternative (11). The plots of ( vs. pH obtained for [Aspl,Val5]angiotensin II a proton, contrary to what we observed for the other three and the three analogs under study are shown in Fig. 2. Excellent peptides, or any other peptide of our experience, shifts down- agreement exists between the curves of [Aspl,Val5]angiotensin field during the deprotonation of the -COOH group. Fur- II and those published for [Asnl,Val5]angiotensin 11 (13). We thermore, this signal reflects the phenolic titration of Tyr4, the also note the parallelism between the curves of [Aspl,Val5]- side chain that is four residues distant, in contrast to the signals angiotensin II and its inhibitor [Sarl,11e8]angiotensin II; the of 11e5, in which the tyrosine titration is not visible. parallelism, together with the results presented above, removes any doubt as to the analogs' conformational identity. DISCUSSION The most interesting items of the titration curves are: Previous studies on the conformation of angiotensin II indicated Beyond the effect of titration (-COOH, imidazolium, that the residues in the central segment of this peptide, Tyr- -NH3+, and phenol group) on the protons directly concerned, Ile-His, align themselves in a nearly extended structure, whereas we also observe the following long-range influences in angio- residues at the COOH terminus, His-Pro-Phe, are arranged in tensin II and in [SarlIle8]angiotensin II: the imidazolium ti- a loop (14). Further, the side chains of tyrosine and histidine tration (apparent pK = 6.50) is reflected in the titration curves are arranged on the same side and are allowed to rotate almost of the a proton of Tyr4 of the ortho- and meta- protons of the without constraint. The degree of mutual influence between tyrosine side chain, and of the high-field ( proton of Pro7. the imidazole (position 5) and the phenol group (position 4) Conversely, the phenol/phenolate titration is affected by the gives a measure of both the speed of their rotation and the local side chain in position 5, the CGH, the C2H, and the C4H protons backbone geometry. of His6 and the same ( proton of three-times-removed pro- [Sarl,Ile8]Angiotensin II, which is a competitive inhibitor of line. angiotensin II, gave spectroscopic data practically identical to This contrasts with the titration curves of N-methylated those of angiotensin II. Slight differences, if any, were due to analogs. In [Sarl,MeTyr4,Ile8]angiotensin II the observed shift replacement of in position 8 with isoleucine or variation due to the imidazole titration is restricted to the-pro- substitution of in position 1 with sarcosine. These tons of the histidine residue and the CcH of tyrosine. The ortho- results suggest that the backbone and side-chain conformation and meta- protons of the tyrosine side chain and the ( proton of the inhibitor molecule resembles that of the active hormone of do not reflect this effect. Nor do the C2H and C4H and thus mimics the parent hormone at the membrane receptor. histidine side chain signals respond to the phenol titration. Only In other words, the peptide conformation (and this includes the a proton of histidine and, to a diminished extent, the 6 dynamic features such as conformational "flexibility and proton of proline undergo correlated shifts. Even stronger adaptability") may be playing an important role in recognition modifications in the titration curves, compared to [Sar',1le8]- process, and, if the conformation is altered severely, binding angiotensin II, are observed with [Sar1,MeIle5,Ile8]angiotensin will not occur. Thus, it further strengthens our confidence that II. Comparing the effect of histidine and tyrosine titrations to alterations in peptide structure that produce a destruction of what we saw in the other peptides, we notice the conspicuous biological effect also produce marked conformational changes absence of mutual influence; the tyrosine signals of the (major) as observed by spectroscopic methods. trans conformation (a,, ortho-, and meta-) do not indicate the Introduction of a methyl group on a peptide nitrogen in histidine pK, nor do any histidine protons show the phenol ti- position 4 or 5 in [Sarl,Ile8]angiotensin II caused a "kink" in the tration. (The ortho- and meta- resonances of the minor cis- central core of the peptide that resulted in perturbation of the form-which can be observed because of the cis-trans isom- side-chain conformation, though this effect was not of the same erism of the tertiary Tyr4-MeIle5 peptide bond, does reflect the magnitude for position 4 as for position 5. This added kink also histidine titration, and to quite a surprising degree.) The his- distorts the bend observed in the COOH-terminal tripeptide. tidine titration, however, is strongly reflected by the Pro7 res- The rotational restriction of the tyrosine side chain is less pro- idue in this peptide as the a proton and the ( protons are dis- nounced in [Sarl,MeTyr4,Ile8Jangiotensin II than in [Sarl,- tinctly shifted between pH 5 and 7. MeIle5,Ile8]angiotensin II (Table 1). Yet the most striking deviation in these titration curves is Conformation studies with [Sarl,MeTyr4,Ile8]angiotensin II evident for Ile8 in [Sarl,MeIle5,Ile8]angiotensin II, in which the indicated that: (i) only the chemical shifts of close-by residues Downloaded by guest on September 25, 2021 86 Biochemistry: Piriou et al. Proc. Natl. Acad. Sci. USA 77 (1980) are at variance with those found in [Sarl,Ile8jangiotensin II; and tagonist in aqueous solution must be a standard property of (ii) The mutual influence of Tyr4 and His6 is still reflected, analogues possessing the same biological activity. These studies though to a diminished extent. In contrast, with [Sar',Me- may prove useful in designing potent and long-lasting ana- Ile5,Ile8]angiotensin II even the a proton of the COOH-terminal logues. isoleucine residue was strongly affected and, in addition, we observed: (i) a practically total rotation restriction of the side This work was supported in part by National Science Foundation chains of tyrosine, isoleucine, and histidine residues (compare Grant BMS72-02556A01 and National Institutes of Health Grant CD signal intensity and coupling constants); (ii) the absence of HL-6835. the usual Tyr4-His6 interdependence as evidenced by CD and 1. Khosla, M. C., Smeby, R. R. & Bumpus, F. M. (1973) in Handbook NMR titration curves; (iii) a modified orientation of histidine of Experimental Pharmacology, eds. Page, I. H. & Bumpus, F. toward proline at acid pH and an enhanced titration effect of M. (Springer, Berlin), Vol. 37, pp. 126-161. histidine on proline signals; and (iv) a strong mutual influence 2. Regoli, D., Park, W. K. & Rioux, F. (1974) Pharmacol. Rev. 26, between the Tyr4 and Ile8 residues as reflected by the titration 69-123. curves of the carboxyl group seen on the Tyr4 signal (Fig. 1) and 3. Khosla, M. C., Hall, M. M., Smeby, R. R. & Bumpus, F. M. (1974) the phenol titration curve seen on the Ile8 a-proton signal (Fig. J. Med. Chem. 17,431-433. 2). It may be pointed out that interaction, as 4. Khosla, M. C., Munoz-Ramirez, H., Hall, M. M., Smeby, R. R., observed by CD Khairallah, P. A., Bumpus, F. M. & Peach, M. J. (1976) J. Med. titration curves, also persists in [Ile8]angiotensin II and [Sarl,- Chem. 19, 244-250. Ile8]angiotensin II but to a lesser extent. The conformational 5. Tonelli, E. E. (1976) Biopolymers 15, 1615-1622. significance of this influence may be interpreted as follows: the 6. Patel, D. J. & Tonelli, A. E. (1976) Biopolymers 15, 1623- carboxyl group must be in the vicinity of the rotating tyrosine 1635. ring, and this is possible only if the proper folding of the 7. Lintner, K., Fermandjian, S., Regoli, D. & Barabe, J. (1977) Eur. COOH-terminal tripeptide occurs. J. Biochem. 61,395-401. Thus, these results suggest: 8. Lintner, K., Fermandjian, S., Fromageot, P., Khosla, M. C., (i) For maximum agonistic or antagonistic activity the Smeby, R. R. & Bumpus, F. M. (1977) Biochemistry 16, 806- conformation of the analogue should resemble that of the parent 812. 9. Lintner, K., Fermandjian, S., Fromageot, P., Khosla, M. C., hormone so that it can mimic the hormone in recognizing and Smeby, R. R. & Bumpus, F. M. (1975) FEBS Lett. 56, 366- binding with the receptor site on the cell. 369. (ii) Drastic decrease in antagonistic activity obtained with 10. Glickson, J. D., Cunningham, W. D. & Marshall, G. R. (1973) N-methylated.amino acids in positions 4 or 5 in [Sarl,Ile8]- Biochemistry 12, 3684-3692. angiotensin II is perhaps due to rotational restriction of the side 11. Kopple, D. D., Wiley, G. R. & Tauke, R. (1973) Biopolymers 12, chains in positions 4 and 5. 627-636. (iii) The isoleucine residue in position 5 appears to have a 12. IUPAC-IUB Commission on Biochemical Nomenclature (1970) controlling influence in aligning the residues in the central J. Mol. Biol. 52, 1-17. segment (Tyr-Ile-His) in angiotensin II and its potent analogues 13. Fermandkian, S., Piriou, F., Toma, F., Lam-Thanh, H., Lintner, in a nearly K., Vicar, J. & Fromageot, P. (1978) in Proceedings of the Eu- extended form. ropean Conference on NMR on Macromolecules, Sardaigne, (iv) The application of NMR and CD spectroscopy appears May 1978, ed. Conti, F. (Lerici, Sassari, Italy), pp. 229-242. to be of great help in studying peptide conformation-biol6gical 14. Fermandjian, S., Lintner, K., Haar, W., Fromageot, P., Khosla, activity relationships. The titration effects as well as the frac- M. C., Smeby, R. R. & Bumpus, F. M. (1976) in Peptides 1976, tions of rotamer populations characterizing the histidine and ed. Loffet, A. (Editions de l'Universite de Bruxelles, Bruxelles, tyrosine side-chain arrangement in angiotensin II and its an- Belgium), pp. 339-352. Downloaded by guest on September 25, 2021