Proc. Nati. Acad. Sci. USAVol. 77, No. 1, pp. 82-86, January 1980Biochemistry

Amino acid side chain conformation in angiotensin II and analogs:Correlated results of circular dichroism and 1H nuclearmagnetic resonance

(peptide hormones/competitive inhibitor/N-methylation/pH titration)

F. PIRIou*, K. LINTNER*, S. FERMANDJIAN*, P. FROMAGEOT*, M. C. KHOSLAt, R. R. SMEBYt, ANDF. 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-Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent an-tagonist of the pressor action of angiotensin II. With a view toincrease half-life in vivo of this peptide, the amino acid residueat position 4 (tyrosine) or position 5 (isoleucine) was replacedwith the corresponding N-methylated residue. This changedrastically reduced the antagonistic properties of this analog.The present work was therefore undertaken to investigate theeffect of N-methylation on overall conformation of these pep-tides and to determine the conformational requirements formaximum agonistic or antagonistic pro rties. Conformationstudies were carried out by circular dichroism and proton nu-clear magnetic resonance spectroscopy in aqueous solution asa function of pH. The results indicated that: (i) angiotensin IIand [1-sarcosine,8-isoleucinelangiotensin II gave practicallyidentical spectroscopic data; and (ii) N-methylation in eitherposition 4 or position 5 resulted in remarkable changes in thepeptide backbone and a severe limitation in rotational freedomof side chains in tyrosine, isoleucine, and histidine residues.However, rotational restriction of the tyrosine side chain wasfound to be less pronounced in [1-sarcosine,4-N-methyltyro-sine,S-isoleucinelangiotensin II than in [1-sarcosine,5-N-methylisoleucine,S-isoleucinejlangiotensin IL Thus, these resultssuggest that: (I) the backbone and side chain structure of a potentangiotensin II antagonist should resemble that of the hormone,angiotensin II, so that it can mimic the hormone in recognizingand binding with the receptor on the cell membrane; and (ii)greater impact of N-methylation in position 5 on the overallconformation of these peptides points to the controlling influ-ence of position 5 (isoleucine) in aligning the residues in thecentral segment (tyrosine-isoleucine-histidine) of angiotensinII and its potent agonist and antagonist analogs in a nearly ex-tended structure. Any change in this arrangement may lead toreduced biological activity.

Recognition of a biologically active molecule by its receptor siteis probably the first event of the hormone-receptor interactionprocess, whereas binding and signal release are subsequentphenomena. Thus, it is likely that during the approach of apeptide to the cell membrane the local environment maygreatly influence the selection of a "recognizable" conformationamong numerous other conformations. It is therefore assumedthat the peptides showing physical properties similar to thoseof the parent hormone, in a given set of experimental conditions(e.g., solvent and pH effects, etc.), may survive the conforma-tional selection procedure during their approach to the cellmembrane. The present work was undertaken to provide evi-dence for this hypothesis, by using angiotensin analogues asmodel peptides.

[Sarl,11e8]Angiotensin II was shown to be a potent antagonistof the pressor action of antiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). However, this and other similar antagonisticpeptides have short half-lives in vivo; the shortness is presum-ably due to rapid degradation by peptidases (for detailed re-views, see refs. 1 and 2). With a view to render these peptidesmore resistant to enzymatic degradation, the residue at position4 (tyrosine) or position 5 (isoleucine) was replaced with thecorresponding N-methylated amino acid. The analogs thussynthesized, [Sar',MeTyr4,I1e81- and [Sar1,MeIle5,Ile8jangio-tensin II showed drastically reduced antagonistic properties (3,4). It is possible that the reduced activity due to N-methylationmay be a consequence of either modification of backboneconformation or rotational restriction of the side chain, or both.It has been shown that N-methylation of a single amino acidresidue in a peptide chain restricts the number of conformationsnot only of the N-methylated residues but also of the residuepreceding it (5, 6). Further, N-methylation may also affect thelowest energy conformation of the involved fragment. There-fore, the conformations of the above analogs and those of an-giotensin II and [Sar',Ile8Jangiotensin II in water solution wereexamined as a function of pH. Circular dichroism (CD) andproton NMR spectroscopy were used to obtain informationabout the side-chain conformations, especially with regard tothose residues that are important for biological activity, namely,tyrosine and histidine.

MATERIALS AND METHODSThe synthesis and purification of the peptides have been de-scribed (3, 4). CD spectra were recorded on a Dichrographmodel Mark III (Jobin Yvon). Peptide concentrations weremade to about 0.5 mM and were determined by ultravioletabsorbance (E275 = 1350 M-' cm-' at pH 5.8). The previouslydescribed method of pH titration was used without modifica-tion (7). Theoretical titration curves were calculated with thehelp of a computer program "Titrage" kindly made availableby M. Gingold. Results are expressed in molar ellipticity [0I =3300 Ae, in units of ocm2/dmol.

'H NMR spectra were recorded at 250 MHz on a CAMECATSN 250 instrument in the Fourier transform mode. Thepeptide concentration in 2H20 was 35 mM. The internal ref-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,Ile8langiotensinII, for which 40'C was necessary to shift the HO2H peak).

Abbreviation: CD, circular dichroism.

82

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Proc. Natl. Acad. Sci. USA 77 (1980) 83

RESULTSCDMarked differences were observed in the CD spectra of the fourpeptides dissolved in water at pH 5.8, recorded in the aromaticregion (320-250 nm). The sign and the intensity of the tyrosinesignal at the initial pH value (5.8) are given in Fig. 1, in whichthe values for [Sar',MeIle5,Ile8]angiotensin II (+450) and[Sarl,MeTyr4,Ile8langiotensin II (-850) are noteworthy. Signalintensity, which in tyrosine model compounds and previouslystudied angiotensin II analogs was found to be in the +300range, is of limited diagnostic value and indicates at best theamount of local asymmetry or hindered rotational freedom ofthe chromophore. On the other hand, the comparison of titra-tion curves obtained for different peptides yields precisestructural information (8). This is also true for the peptidespresented here, for which we shall discuss the three zones of pHshown in Fig. 1.

Carboxyl Titration. The intensity of the 'Lb signal of tyrosinein angiotensin II is sensitive to the titration of one or morecarboxyl groups (Fig. 1A) (8). It had been observed that theslope of ihe titration curve is dependent on the presence orabsence of the f3-carboxyl group in position 1: it is positive in[Aspl]angiotensin II peptides but negative in [Asn']angiotensinII, [Me2Glyl]angiotensin II, and des-Aspl-angiotensin II, etc.(8). Whereas the possibility of conformational changes in thebackbone structure during COOH titration or an indirecteffect via the histidine side chain cannot be denied, the relativedistance and orientation of the carboxyl group toward the ty-rosine side chain is likely to be the main factor leading to theobserved effects. In the event of similar backbone structure, thisdistance is primarily determined by the time-averaged orien-tations of the tyrosine side chain. In this light, the curves shownin Fig. 1A are strongly significant. The slope for angiotensinII is positive (Aspl), the one for [Sarl,11le8]angiotensin II isnegative (a COOH, COOH-terminal). In [Sar1,MeIle5,Ile8]-angiotensin II the carboxyl titration has an enormous impacton the CD signal: A[OJ = -230 compared to A[6] = -20for [Sarl;Ile8]angiotensin II. The tyrosine signal in [Sarl,MeTyr4,Ile8]angiotensin II, on the other hand, is removed fromany influence of the carboxyl titration.

pH

FIG. 1. Titration curves. For A and B, ellipticity was measuredat 275 nm; for C, 293 nm. A, Angiotensin II; *, [Sarl,l1e8]angiotensinII; o, [Sar',MeTyr4, Ile8]angiotensin II; *, [Sar',MeIle5,Ile8Jangio-tensin II.

Histidine Titration. Arguments based on data from a largenumber of analogs studied (9) have shown that the inversionof the tyrosine signal in angiotensin II (Fig. lB) is due to thetitration of the close-up histidine side chain. The amplitude ofthe titratiop curve observed for a given peptide is a function ofits conformational characteristics especially with respect to thecentral residues 3 to 7 of angiotensin 11 (8). The curves in Fig.1B follow this pattern. Angiotensin II and its potent inhibitor,[Sarl,Ile8]angiotensin II, have curves that are almost superim-posable (A[0J = -450), which confirms the titration behaviorof the less-modified inhibitor [Ile8]angiotensin II (8). A markedreduction in the side-chain-side-chain influence is observedfor [Sar',MeTyr4;Ile8]angiotensin II (A[O] = -200). The in-tensity of the 'Lb band in this peptide hints at hindered rotationof the tyrosine side chain, which could account for an increasedtime-averaged distance between the two aromatic side chainstyrosine and histidine. Again, these factors are possibly tied inwith as yet unquantifiable changes in backbone conformation.The titration curve for [Sarl,MeIle5,le8]angiotensin II, on theother hand, is flat in this pH region; the respective orientationof the tyrosine arnd histidine side chains and the distance be-tween them has clearly been upset by the N-methylation andthe concomitant changes of dihedral angles 4 and i/' along thecentral stretch of the backbone.

Phenol Titratidn. It is only between pH 9 and pH 12 that wetitrate the chromophore of the tyrosine side-chain proper. Thechange in electronic structure causes the well-documented shiftof the 'Lb band from 275 nm to 293 nm; in almost all cases sofar published in the literature or observed in our laboratory(scores of the most diverse tyrosine-containing peptides), this'Lb dichroic band of Tyr-0- was observed to be positive withellipticities between 300 and 800 units. The curves for angio-tensin II, .[Sar',1le8]angiotensin II, and [Sar',MeIle5,Ile8]an-giotensin II (Fig. 1C) are therefore quite normal. The surprisinghigh intensity negative band found for [Sarl,MeTyr4,Ile8]-angiotensin II must then be seen as a sign of distinctly differentside-chain orientation vis a vis its environment.'H NMRChemical Shifts. The resonances in the spectrum of angio-

tensin II were assigned on the basis of double resonance ex-periments and pH titration effects; the assignments agree wellwith data published previously (10). They serve as basis for thesignal assignment in the analogs discussed below. Chemicalshifts of the resonances in the competitive inhibitor [Sar',1le8]-angiotensin II are quite similar to those found in angiotensinII, with the exception of those for Sarl and Ile8, of course. TheN-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; thischange also has profound effects on the NMR spectra: Theresonances of the a protons of the N-methylated residue andthose preceding it in the sequence are considerably shifted tolower 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 removedresidues are not affected compared to [Sarl,Ile8Jangiotensin II,except for the a proton of the COOH-terminal isoleucine suchas in [SarlMeIle5,11e8]angiotensin II, for which a 0.39-ppmdownfield shift was registered. Whereas the immediate causesfor the observed chemical shift variations between these pep-tides may lie in variations of electronic density and localmodifications of steric hindrance due to the effects of -CH3,other factors responsible for the observations will be discussedbelow.

Coupling Constants. Whereas the spectrum of [Sar',Ile8J-angiotensin II at 250 MHz allows the various /-proton signals

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Proc. Natl. Acad. Sc. USA 77 (1980)

1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 7 9 11pH

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, CoHIle5; *, C5H Pro7; A, C6H Pro7.

(AB parts) to be observed clearly, signal overlapping in an-

giotensin II-e.g., in [Sar',MeTyr4,Ile8]angiotensin II and[Sar',MeIle5,Ile8]angiotensin II-precludes exact analysis ofthis region. The 1H NMR spectrum of angiotensin II obtainedat 360 MHz, however, renders complete analysis of the ABXpatterns possible (unpublished results). For the other twopeptides, coupling constants were measured on the a-protonsignals. Computer calculations (program "ITRCAL," NicoletInstrument Corporation, Madison, WI) on the 360 Mhz and250-MHz spectra show the results obtained from measuring the3JC-H-GH coupling constants on the a-proton signals to be valid.These coupling constants are indicators of side-chain orientationand are usually used to determine rotamer populations or thedirect value of the Xi dihedral angle read from a suitableKarplus curve (11). The data observed for angiotensin II andits analogs are listed in Table 1 together with the respectiverotamer fractions. Assignment of rotamers I and II was cori-firmed by 13C NMR spectroscopy of uniformly '3C-enrichedresidues in angiotensin II peptides (13). For angiotensin II, therotamer distribution varies with the nature of the side chain,but not significantly with pH [rotamer I predominated in Aspl,Tyr4, and Phe8; rotamer II in Val5 and Ile5; rotamers I and IIare preponderant in His6 (Table 1)]. The same values are foundfor the side chains in [SarlIle8langiotensin II, confirming theconformational homogeneity with angiotensin II already noted

with the chemical shifts. On the other hand, the NMR param-

eters of [Sar',MeTyr4,I1e8]angiotensin II are different from thoseof [Sarl,l1e8]angiotensin II principally with respect to the ty-rosine side chain, but also with respect to Ile5 and, more sur-

prisingly when we consider the chemical shift effects discussedabove, the twice-removed His6 residue. The most drastic effects,however, are observed for [Sarl,MeIle5,Ile8jangiotensin II, inwhich the side-chain orientations of Tyr4, IVe5, and His6 are stillmore strongly perturbed. The coupled constant of 11.3 Hz forlie5 translates into a rotamer II fraction of 0.8 (X = 180°) andindicates almost total absence of rotation. For tyrosine andhistidine it seems reasonable, too, to interpret the couplingconstants (10.0 and 4.8 Hz) in terms of a unique X angle of 0°or 120°, even though this would correspond to the eclipsedconformation. Steric factors originating in the N-methyl groupwould easily account for this, though. Clearly, the conforma-tional properties of angiotensin II show a higher sensitivity toN-methylation in position 5 than in position 4.

Titration Curves. Proton chemical shifts are sensitive tocharge variations in their proximity and thus to pH titrationsof ionizable groups, if they are close because of chemicalstructure or spatial/directional preferred orientation. The pHtitration curves of proton resonance signals constitute a powerfulmeans to assign signals to specific residues and to determinespatial proximity between atoms or groups that are structurallydistant.

N

I 1E'0

84 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 IIResidue 3JH--HO RI RII 3JH"-Ht RI RII 3JHI-HI RI R11 3JH"-HO RI RII

Asp 8.0 0.495.0 0.21

Arg 7.0 0.40 7.0 0.40 7.0 0.40 7.0 0.407.0 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 0.49Tyr 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 0.20Ile 8.0 0.49 8.6 0.54 8.0 0.49 11.3 0.80His 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 0.20Pro 8.5 8.8 8.6 8.8

5.0 5.8 5.5 4.7Ile or 8.0 0.49 6.0 0.31 6.0 0.31 6.5 0.35Phe 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 IIand the three analogs under study are shown in Fig. 2. Excellentagreement exists between the curves of [Aspl,Val5]angiotensinII and those published for [Asnl,Val5]angiotensin 11 (13). Wealso note the parallelism between the curves of [Aspl,Val5]-angiotensin II and its inhibitor [Sarl,11e8]angiotensin II; theparallelism, together with the results presented above, removesany doubt as to the analogs' conformational identity.The most interesting items of the titration curves are:Beyond the effect of titration (-COOH, imidazolium,

-NH3+, and phenol group) on the protons directly concerned,we also observe the following long-range influences in angio-tensin II and in [SarlIle8]angiotensin II: the imidazolium ti-tration (apparent pK = 6.50) is reflected in the titration curvesof the a proton of Tyr4 of the ortho- and meta- protons of thetyrosine side chain, and of the high-field ( proton of Pro7.Conversely, the phenol/phenolate titration is affected by theside chain in position 5, the CGH, the C2H, and the C4H protonsof His6 and the same ( proton of three-times-removed pro-line.

This contrasts with the titration curves of N-methylatedanalogs. In [Sarl,MeTyr4,Ile8]angiotensin II the observed shiftvariation due to the imidazole titration is restricted to the-pro-tons of the histidine residue and the CcH of tyrosine. The ortho-and meta- protons of the tyrosine side chain and the ( protonof proline do not reflect this effect. Nor do the C2H and C4Hhistidine side chain signals respond to the phenol titration. Onlythe a proton of histidine and, to a diminished extent, the 6proton of proline undergo correlated shifts. Even strongermodifications in the titration curves, compared to [Sar',1le8]-angiotensin II, are observed with [Sar1,MeIle5,Ile8]angiotensinII. Comparing the effect of histidine and tyrosine titrations towhat we saw in the other peptides, we notice the conspicuousabsence of mutual influence; the tyrosine signals of the (major)trans conformation (a,, ortho-, and meta-) do not indicate thehistidine pK, nor do any histidine protons show the phenol ti-tration. (The ortho- and meta- resonances of the minor cis-form-which can be observed because of the cis-trans isom-erism of the tertiary Tyr4-MeIle5 peptide bond, does reflect thehistidine titration, and to quite a surprising degree.) The his-tidine titration, however, is strongly reflected by the Pro7 res-idue in this peptide as the a proton and the ( protons are dis-tinctly shifted between pH 5 and 7.

Yet the most striking deviation in these titration curves isevident for Ile8 in [Sarl,MeIle5,Ile8]angiotensin II, in which the

a proton, contrary to what we observed for the other threepeptides, or any other peptide of our experience, shifts down-field during the deprotonation of the -COOH group. Fur-thermore, this signal reflects the phenolic titration of Tyr4, theside chain that is four residues distant, in contrast to the signalsof 11e5, in which the tyrosine titration is not visible.

DISCUSSIONPrevious studies on the conformation of angiotensin II indicatedthat the residues in the central segment of this peptide, Tyr-Ile-His, align themselves in a nearly extended structure, whereasresidues at the COOH terminus, His-Pro-Phe, are arranged ina loop (14). Further, the side chains of tyrosine and histidineare arranged on the same side and are allowed to rotate almostwithout constraint. The degree of mutual influence betweenthe imidazole (position 5) and the phenol group (position 4)gives a measure of both the speed of their rotation and the localbackbone geometry.

[Sarl,Ile8]Angiotensin II, which is a competitive inhibitor ofangiotensin II, gave spectroscopic data practically identical tothose of angiotensin II. Slight differences, if any, were due toreplacement of phenylalanine in position 8 with isoleucine orsubstitution of aspartic acid in position 1 with sarcosine. Theseresults suggest that the backbone and side-chain conformationof the inhibitor molecule resembles that of the active hormoneand thus mimics the parent hormone at the membrane receptor.In other words, the peptide conformation (and this includesdynamic features such as conformational "flexibility andadaptability") may be playing an important role in recognitionprocess, and, if the conformation is altered severely, bindingwill not occur. Thus, it further strengthens our confidence thatalterations in peptide structure that produce a destruction ofbiological effect also produce marked conformational changesas observed by spectroscopic methods.

Introduction of a methyl group on a peptide nitrogen inposition 4 or 5 in [Sarl,Ile8]angiotensin II caused a "kink" in thecentral core of the peptide that resulted in perturbation of theside-chain conformation, though this effect was not of the samemagnitude for position 4 as for position 5. This added kink alsodistorts the bend observed in the COOH-terminal tripeptide.The rotational restriction of the tyrosine side chain is less pro-nounced in [Sarl,MeTyr4,Ile8Jangiotensin II than in [Sarl,-MeIle5,Ile8]angiotensin II (Table 1).

Conformation studies with [Sarl,MeTyr4,Ile8]angiotensin IIindicated that: (i) only the chemical shifts of close-by residues

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Proc. Natl. Acad. Sci. USA 77 (1980)

are at variance with those found in [Sarl,Ile8jangiotensin II; and(ii) The mutual influence of Tyr4 and His6 is still reflected,though to a diminished extent. In contrast, with [Sar',Me-Ile5,Ile8]angiotensin II even the a proton of the COOH-terminalisoleucine residue was strongly affected and, in addition, weobserved: (i) a practically total rotation restriction of the sidechains of tyrosine, isoleucine, and histidine residues (compareCD signal intensity and coupling constants); (ii) the absence ofthe usual Tyr4-His6 interdependence as evidenced by CD andNMR titration curves; (iii) a modified orientation of histidinetoward proline at acid pH and an enhanced titration effect ofhistidine on proline signals; and (iv) a strong mutual influencebetween the Tyr4 and Ile8 residues as reflected by the titrationcurves of the carboxyl group seen on the Tyr4 signal (Fig. 1) andthe phenol titration curve seen on the Ile8 a-proton signal (Fig.2). It may be pointed out that interaction, as observed by CDtitration curves, also persists in [Ile8]angiotensin II and [Sarl,-Ile8]angiotensin II but to a lesser extent. The conformationalsignificance of this influence may be interpreted as follows: thecarboxyl group must be in the vicinity of the rotating tyrosinering, and this is possible only if the proper folding of theCOOH-terminal tripeptide occurs.

Thus, these results suggest:(i) For maximum agonistic or antagonistic activity the

conformation of the analogue should resemble that of the parenthormone so that it can mimic the hormone in recognizing andbinding with the receptor site on the cell.

(ii) Drastic decrease in antagonistic activity obtained withN-methylated.amino acids in positions 4 or 5 in [Sarl,Ile8]-angiotensin II is perhaps due to rotational restriction of the sidechains in positions 4 and 5.

(iii) The isoleucine residue in position 5 appears to have acontrolling influence in aligning the residues in the centralsegment (Tyr-Ile-His) in angiotensin II and its potent analoguesin a nearly extended form.

(iv) The application of NMR and CD spectroscopy appearsto be of great help in studying peptide conformation-biol6gicalactivity relationships. The titration effects as well as the frac-tions of rotamer populations characterizing the histidine andtyrosine side-chain arrangement in angiotensin II and its an-

tagonist in aqueous solution must be a standard property ofanalogues possessing the same biological activity. These studiesmay prove useful in designing potent and long-lasting ana-logues.

This work was supported in part by National Science FoundationGrant BMS72-02556A01 and National Institutes of Health GrantHL-6835.

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86 Biochemistry: Piriou et al.