Correlation of the biological activity and solution conformation of [Asp1,Ile5]- and [Phe4,Tyr8]angiotensin II.

Abstract

Angiotensin I is known to undergo a reversible conformational transition and a change in potency in rat uterus in vitro with pK -6.5. We have shown by carbon-13 NMR that the conformational transition involves all-trans to partly cis isomerization of the His6-Pro7 peptide bond. Isomerization from all-trans at pH 6.8 to "16% cis at pH 8.0 is therefore correlated with a 10-fold increase in biological activity for [Asp',IJeWJ angiotensin II in rat uterus in vitro. Isomerization from all-trans at pH 6.8 to "16% cis at pH 8.0 in the competitive inhibitor [Phe4,Tyr8Jangiotensin II is correlated with exhibition of virtually no agonist activity at low pH to full agonist activity at high pH. An angiotensin II conformation with Pro7 in the cis form may therefore be the conformation with maximal binding or biological activity at the cellular receptor.Our understanding of the physical and chemical basis for the biological activity of angiotensin II has progressed along two largely independent pathways. The investigation of the biological activity of a large number of analogs (summarized in refs. 1-3) has clarified the role that various residues or groups of residues play in that activity. The investigation of the solution conformation of angiotensin II and of various analogs has made use of most biophysical methods available (summarized in refs. 4 and 5), and several models for the solution conformation have been proposed (5-9). Although most investigators agree that a compact and more or less rigid conformation does exist, no agreement on a definite model has been reached. We suggest that the biological potency of "native" angiotensin II § and of [Phe4,Tyr8]angiotensin II as a function of pH may be related to the amount of cis configuration of Pro7 present in solution. The [Phe4,Tyr8] analog of angiotensin II has been observed to be an antagonist at low pH and an agonist above pH 7 (11).The present study is based on our earlier observations (10) that at low pH in aqueous solution angiotensin II is present in a conformation having proline in the trans configuration and above pH 6.5 two conformations are observed by nuclear magnetic resonance (NMR), an abundant conformation (88%) with proline in the trans configuration and a minor conformation (12%) with proline in the cis configuration. The evidence for this conclusion was that the carbon-13 NMR spectrum at and above neutral pH of the COOH-terminal hexapeptide fragment of [Val5]angiotensin II showed a resonance of the required intensity at the position expected for proline C-'y when proline is in the cis configuration. The pH dependence of the conformation of angiotensin observed by NMR is well documented (4,5,(12)(13)(14) and the possible relationship of this conformational change with pK -6.5 to myotropic activity has been discussed (5, 13). We show here by '3C NMR that both [Asp1,Tyr8]angiotensin II isomerize from alltrans at acidic p2H to partly cis at alkaline p2H and that this isomerization is reported by the His6 C-2 (and C-4) protons. Other conformational...

s-Cis and s-trans isomerism of the His-Pro peptide bond in angiotensin and Thyroliberin analogs

“…The cis-trans exchange rates have not been determined for angiotensin or TRH peptides. However, the cis and trans C-2 proton resonances in [Asp1,lie5]-angiotensin II at pD 9.1 do not coalesce at temperatures up to 60 °C (Bleich et al, 1978), indicating an exchange rate not likely to be faster than that of His-Pro.…”

“…The percentage of the cis isomer measured by the proline Ccarbon is 50% at pD 7.4. tinguish between the complete absence of the trans to cis isomerization and the occurrence of the isomerization without the reporting of it by the His side chain. The maximum percentage of s-cis isomer reported by the His C-2 proton in [3-MeHis6]-angiotensin is ~25% compared with 16% for [Asp1,lie5]-angiotensin and 17% for the [Phe4,Tyr8]-angiotensin II (Bleich et al, 1978). Table III also shows the pD dependence of the proton chemical shifts of the His C-2 and C-4 protons for A"-acetyT, [Sar'.Ala8]-, and [homoHis6]angiotensin II.…”

“…The pMs of the isomerizations were estimated from a graph of the percentage of cis isomer as a function of pD. The assignment of the s-cis and s-trans His C-2 and C-4 protons is based on our previous studies (Galardy et al" 1976;Bleich et al, 1978) and on the and l3C NMR spectra of His-Pro and TRH. The assignment of s-cis and s-trans isomers in His-Pro and TRH was based on and 13C NMR of the proline side chain.…”

“…The pK of this isomerization is about 6.5 in D20 and the histidine C-2 and C-4 protons report the isomerization (Bleich et al, 1978;Galardy et al, 1976). Therefore, the change in the conformation of angiotensin around neutral pH Marshall et al, 1974) involves, at least in part, all-s-trans to partly s-cis isomerization of proline.…”

The rate of s‐cis/s‐trans isomerization in angiotensin II is at least 70‐fold greater than in His‐Pro and is not rate limiting in receptor binding

“…at 22.5", of the same magnitude as the relaxation time for the slow phase of folding of a number of small proteins (1). 1) upon titration from low to physiological pH (7). angiotensin (3, 4), oxytocin (9, thyroliberin ( 6 ) and luliberin (6).…”