Eur. J. Biochem. 231, 370-380 (1995) 0 FEBS 1995

The basic subdomain of the c-Jun oncoprotein A joint CD, Fourier-transform infrared and NMR study

Daniel KREBS ', Benaniar DAHMANI', SaYd EL ANTRI', Monique MONNOT', Odile CONVERT*, Olivier MAUFFRET', FredCric TROALEN and Serge FERMANDJIAN'

' Dkpartement de Biologie Structurale, URA 147 CNRS, Institut Gustave Roussy, Villejuif, France * Chimie Organique Structurale, Universitk Pierre et Marie Curie, ERS 073 CNRS, Paris, France

Service d'Immunologie Moleculaire, Institut Gustave Roussy, Villejuif, France

(Received 3 MarcM24 April 1995) - EJB 95 034213

The structural properties of the basic subdomain of the basic zipper (bZIP) protein c-Jun were exam- ined by joint means of 'H-NMR, CD and Fourier-transform infrared (FTIR) spectroscopies. The basic subdomain (residues 252 -281 in c-Jun) is responsible for sequence-specific recognition of DNA. A modified basic subdomain bSD (residues 1-35) and its N-terminal part and C-terminal part fragments (NP, residues 1-19; and, CP, residues 16-35) were prepared by solid-phase synthesis and purified by HPLC. In aqueous solution, in the absence of DNA, bSD behaved mostly as an unstructured peptide characterized by only 5% a helix. However, upon mixing bSD and a specific DNA fragment, i.e. a CRE(cAMP-responsive element)-containing hexadecanucleotide, the a helix was stabilized to an extent of 20% at 20°C or 35% at 2°C. At the same time, no significant change could be detected in the DNA spectra. Addition of trifluoroethanol to an aqueous bSD sample resulted in an increase of the a-helix content so that about 60% of a helix was found at a ratio of 75% trifluoroethanol (20°C). These effects were reflected in both CD and FTIR measurements. Changes shown by the CD spectra during the process suggested a mechanism dominated by a two-state helixhnordered transition. NMR data, namely a, chemi- cal shifts, NOE cross-peaks and N, temperature coefficients provided indications for extended or nascent helix structures within four short stretches dispersed along the sequence for c Jun bSD, contrasting with the unique and continuous stretch reported for Gcn4 (yeast general control protein 4) bSD in aqueous solution. Trifluoroethanol stabilized the a-helix structure mainly at these four sites. The malleability of the basic subdomain of c Jun was emphasized in relation to its ability to fit the DNA helix in adopting an a-helix structure. The complex formation apparently requires substantial conformational change from the peptide and only little from the oligonucleotide.

Keywords. c-Jun ; peptide ; conformation; interactions ; spectroscopy.

The Jun oncoproteins belong to the class of basic zipper tran- scription factors characterized by an N-terminal DNA-binding subdomain rich in basic residues (basic, b) and an adjacent leu- cine-zipper-dimerization subdomain (leucine zipper, ZIP; for a review see [l]). Jun proteins are components of the AP-1 tran- scriptional regulators and are allowed to form homodimers and also heterodimers with various members of the AP-1 family [2]. The resulting dimers bind to the 7 bp consensus sequence TGACTCA (12-0-tetradecanoylphorbol-13-acetate-responsive element). Jun proteins can also dimerize with partners of the CAMP-responsive-element-binding (CREB) proteins family, to bind with a preferential affinity to the 8-bp consensus sequence TGACGTCA (CAMP-responsive element, CRE).

The crystal structures of Gcn4 (yeast general control protein 4) and c-Jun-c-Fos complexed to DNA have been recently re-

Correspondence ro S . Fermandjian, Departement de Biologie Struc- turale, Centre National de la Recherche Scientifique Unite 147, Institut Gustave Roussy, F-94805 Villejuif Cedex, France

Abbreviations. bp, base pair; bSD, basic sub-domain; CP, C-terminal part of bSD; CRE, CAMP-responsive element; CREB, CRE binding; DQF-COSY, double-quantum-filtered correlation spectroscopy; FTIR, Fourier-transform infrared spectroscopy ; Gcn4, general control protein 4 of yeast; HOHAHA, homonuclear Hartman-Hahn experiment ; NOESY, nuclear Overhauser effect spectroscopy; NP, N-terminal part of bSD ; TOCSY, total correlation spectroscopy.

ported [3-51. Gcn4, a yeast basic zipper regulatory protein, is struclurally similar to the Jun oncoproteins [6] and both proteins recognize the same DNA sequences A P K R E to regulate tran- scription [7, 81. The entire basic zipper domain of c-Jun and Gcn4 show an unintermpted a-helix conformation and the a he- lix of the basic subdomain accommodated without noticeable deformation within the DNA major groove [3-51. In the com- plexes of Gcn4 and c-Jun-c-Fos with DNA, base-specific con- tacts between one basic region a helix and a DNA half-site con- cern the same conserved amino acid side-chains [3-51.

Despite the apparent simplicity of interactions authorized by the adjustment of their short a helix within the DNA major groove, the basic zipper proteins offer a remarkable diversity of DNA-binding specificities. It is noticeable, that in its crystal complexes with AP-1 and CRE, the Gcn4 basic zipper peptide shows both similar a-helix conformation and similar interactions with DNA [3, 41, while the half-site spacing is not equivalent in AP-1 and CRE. The question was whether the basic region helix or the DNA double helix or both, were flexible enough to facili- tate the best contacts between the interacting groups in both the AP-1 and the CRE complexes. Some degree of malleability in the central CpG step of CRE has been already shown by us through our joint NMR and energy-minimization study per- formed on a CRE-containing dodecamer [9]. However, the pep-

Krebs et al. ( E m J. Biochem. 231) 371

tide partner, i.e. the basic subdomain of Gcn4, has been shown to display a random secondary structure in aqueous solution [lo-121 although it can specifically interact with the correct target sequences [13, 141 through stabilization into an a helix with, however, reduced affinity relative to the entire protein.

The above data provided the motivation of this NMR, CD and Fourier-transform infrared (FTIR) combined work on the c-Jun basic subdomain peptide in aqueous and trifluoroethanol solutions. c-Jun and Gcn4 belong to the basic zipper protein family and present strong sequence similarities [7]. The aim of the present study was to describe the results of experiments de- signed to evaluate the conformational interchange of cJun bSD. The total assignment of NMR spectra in aqueous solution was achieved by comparing the basic subdomain peptide with two of its fragments. Relatively good correlations were found for conformational effects of trifluoroethanol, between the CD, FTIR and NMR results. Data are consistent with experimental models for helix stabilization upon dissolution into trifluoroetha- no1 for peptides already displaying a nascent helical structure in aqueous solution [15]. The structural variability displayed by c- Jun bSD was compared to that of Gcn4 bSD and discussed in relation with its potency to accomodate an a-helix structure dur- ing its association to DNA.

EXPERIMENTAL PROCEDURES Peptides. The sequence between 252 and 281 of the cJun

basic subdomain (bSD) is shown below together with the de- rived peptides used in this work:

1 10 16 2021 26 30 35

c-Jun (252-281) SQERIKAERK RMRNR IAASK CRKRK LERIA bSD ( 1-35) H-SQERIKAERK RMRNR VAASK SRKRK KERIA KRLYG-OH NP (1-19) H-SQERIKAERK RMRNR VAAS-NH~ CP (16-35) CH~CO-VAASK SRKRK KERIA KRLYG-NH~

bSD derives from the c-Jun segment 252-281 with the following modifications : substitutions in positions 16 (Ile-Val, isomorphous, to facilitate assignment of the two other Ile), 21 (Cys-Ser, induces oncogeny [16,17]) and 26 (Leu-Lys, could stabilize interactions with DNA [ l 81); and, prolongation by five residues including Tyr34 which is useful for determination of concentration by ultraviolet absorption.

Oligonucleotide. The hexadecanucleotide d(GAGA TGACGTCA TCTC) containing the CRE sequence in its center was selected for binding experiments. This segment has been found intact in the somatostatin promoter [19] and represents the minimal size for the study of DNA-protein complexation [12, 201.

Solvents. Analytical grade trifluoroethanol (CF,CH,OH) for CD studies was purchased from Merck-Schuchardt. Deuterated trifluoroethanol (CF,CzH2OZH, 99%) and D,O (99.96 %) for NMR and FTIR studies were purchased from Euriso-top, CEN Saclay, France.

Synthesis. Peptides. The peptides were synthesized by auto- mated solid-phase synthesis according to standard procedures on an Applied Biosystems Model 430 A peptide synthetizer using as resins 4-(oxymethy1)-phenylacetoamidomethyl for the free C- carboxyl peptide, bSD, and, 4-methylbenzhydrylamine for the C-amide peptides, CP and NP. Capping used a mixture of acetic anhydride, N,N-diisopropylethylamine and N-methylpyrroli- done. The immobilized peptides were cleaved and deblocked with low and high hydrofluoric acid (HF) concentrations at 0°C and room temperature, using anisole as scavenger. The products were purified to homogeneity by preparative reverse-phase chro- matography on a Beckman HPLC-126 instrument, using an

Aquapore RP 300 C18 column (220 mmXl0 mm inner diame- ter, 20 p d 3 0 nm). Two solvents were used for HPLC: solvent A, consisting of 0.1% trifluoroacetic acid in H20, and solvent B, consisting of 0.1 % trifluoroacetic acid, 4.9% H20 in acetoni- trile. The crude peptide was dissolved in solvent A before injec- tion. The linear gradient used was from 20% to 80% solvent B in 26 min with a flow rate of 4 d m i n . Fractions containing the major effluent component were lyophilized individually and dissolved in water. For an analytical control, aliquots were chro- matographed by HPLC using an analytical Nucleosil C,, column (250 mmX4.6 mm inner diameter, 5 p d 3 0 nm). The linear gra- dient used was from 20% to 80% solvent B in 77 min, with a flow rate of 1 mVmin.

The identity of each peptide was then assessed by amino acid analysis on a Beckman 6300 amino acid analyzer and, when possible, by Edman degradation (this only works when the N- amino group is free) on an Applied Biosystem 477 A sequencing system. The molecular mass was then verified by electrospray MS (Commissariat ii 1'Energie Atomique, Saclay). Each peptide gave an amino acid composition within 5 % of that expected.

Hexadecanucleotide. This DNA fragment was synthetized using the solid-phase procedure on an Applied Biosystems 380 B automatic apparatus and purified by HPLC on a diethylamino- ethyl-Si column. NMR spectra indicated purity superior to 98 %.

Spectroscopy. CD spectra were recorded on a Jobin-Yvon Mark IV high sensitivity dichrograph linked to a PC micropro- cessor. Optical cells of path-length of 1 mm were placed in a thermostable cell holder. The sample temperature was accurate to within 0.1 "C throughout the experimental range. The concen- tration of the bSD peptide in the sample was determined from absorption measurements at 275 nm of the tyrosine residue (Tyr34 intentionally placed in the peptide) with a value of 1360 M-' . cm-' as molar absorption coefficient at 20°C. Mea- surements were performed in either H20 or in sodiudpotassium phosphate buffer, pH 7, I of 0.1 containing 0.2 mM EDTA, at 20°C and 2°C for peptide concentrations of 2-20X10-5M. The CD signals do not reflect concentration-dependent variations that could originate from molecular associations. The effect of CF,CH,OH titration was examined between 0 and 90 % trifluor- oethanoVH,O (vol. trifluoroethanol addedkotal vol.) at 20°C.

For binding experiments, the CRE-containing hexadeca- nucleotide was at M duplex concentration in sodiudpotas- sium phosphate buffer, pH 7, I of 0.1 containing 0.2 mM EDTA, and with a temperature maintained at either 2°C or 20°C. DNA concentration was calculated using 7800 M-' . cm-'/base as mo- lar absorption coefficient at 260nm, 20°C [Zl]. CD was ex- pressed in units of mean residue ellipticity, [Q], (deg . cmz '

dmol-'). [O] is related to the molar circular dichroism LIE as follows: [ B ] = (3298XA~)/N, where N is the number of residues.

Analysis for prediction of secondary structure from CD spectra was performed by using the method of Yang et al. [22]. Since these predictions are derived from protein secondary struc- tures, they do not seem well suited to peptide structures. Accord- ing to Woody [23], the band at 222 nm is almost exclusively due to the a helix. We therefore used also the relation of Zhong and Johnson [24] : LIsZZZnmX(- 10) to estimate the a-helix content. This appears to be a good alternative to the well known method of Chen et al. [25].

Fourier-transform infrared. Infrared spectra were recorded using a Nicolet 10MX FTIR spectrophotometer equipped with a globar source and a triglycine sulfate (TGS) detector. The spec- trophotometer and sample chamber were continuously purged with dry air. The peptide was placed at 20°C into an infrared cell with CaF, plates and a 50-pm pathlength spacer. For each spectrum, 540 interferograms were collected in the single-beam mode with a resolution of 2 cm-'. To allow proper baseline com-

372 Krebs et al. ( E m J. Biochem. 231)

\ I \ ' \ ,'

I I

pensation, spectra of the buffer solutions were all recorded in the same conditions (cells, pathlength spacer, identical scanning parameters) and substracted from those of peptide solutions.

Solutions of peptide were prepared at a concentration of 11.7 mM in sodiudpotassium phosphate buffer, pH 7, I of 0.1, con- taining 0.2 rriM EDTA and exchanged with D,O. Prior to sample preparation, the trifluoroacetate counterions were replaced with chloride ions by several lyophilisations of the peptide in 0.5 M HCI. This acidic treatment was performed in order to remove trifluoroacetic acid which generates a strong absorption band near 1670 cm-', overlapping with the amide I' absorption band of the peptide [26].

The overlapping components which make up the amide I' band envelop were taken from the Fourier derivative and the deconvoluted spectra [27-291. The amide I' band enhancement resolution was performed assuming a band width of 15 cm-' and a resolution enhancement factor of 2.8 for all spectra. The noise level was evaluated by examining the region above 1700 cm-' in the derivative spectrum.

Quantitative analysis in terms of secondary structures was performed by band decomposition using a least-squares non-lin- ear regression analysis. A linear combination of gaussian and lorentzian bands was used to describe each component. For the final fits, the heights, widths and positions of all the components were varied simultaneously. About 10000 Monte Car10 itera- tions were achieved to estimate the intensity, half-width and po- sition of each component. The band areas obtained by the curve- fitting procedures were estimated up to 2 5 %.

NMR. Proton NMR spectra were recorded at 500 MHz using a Bruker AMX 500 spectrometer equipped with Aspect 3000 and X32 computers, and processed with UXNMR or FELIX softwares.

For one-dimensional and two-dimensional studies, concen- trations for the bSD peptide ranged from 2.1 mM to 8.6mM. No oligomerization effect was detected at these concentrations. The concentrations of NP and CP fragments were 8.2 mM and 10 mM, respectively. Samples of 0.4 ml were prepared in 90% H,0/10% D,O (99.99%) and mixtures of H,O and CF,C2H,02H. The volume of trifluoroethanol was increased from 15 to 25 to 30 to 35 to 50% to 80% on the same sample. For trifluoroethanol titration, 3-trimethylsilyl-propionate was in- cluded as an internal reference standard. In all cases, a small amount of EDTA was added at a final concentration of 1 mM. All solutions were adjusted to pH 3.0, by addition of aliquots of 0.1 M HCI, and uncorrected for isotope and mixed trifluoroetha- nouwater solvents effects. The temperature effects on bSD were measured in aqueous solution from 5 "C to 60 "C by increments of 5"; and, in trifluoroethanol at 5"C, 20°C and 35°C.

NOESY (NOE spectroscopy), TOCSY (total correlation spectroscopy) and DQF-COSY (double-quantum-filtered corre- lation spectroscopy) data sets were collected from 2048 to 4096 real points in t, and from 512 to 1024 t , points using time- proportional phase-incrementation scheme for quadrature detec- tion in the F, dimension [30]. The relaxation delay was set to 1.5 s and spectral widths were 10-12 ppm. Mixing times were varied from 200ms to 600ms in the NOESY experiments. Clean-TOCSY [31] spectra were collected with a 80-120 ms MLEV-17 spin-locking pulse. Solvent suppression was achieved using either presaturation (TOCSY and DQF-COSY) or a jump- return read pulse [32] in NOESY experiments.

Spectra were zero-filled (final data matrix in real points: 204812048 or 409611024 in FJF, dimensions) and subjected to shifted sine bell weighting functions (I716 generally) in both di- mensions before Fourier transformation.

Other methods. Predictions of secondary structures were performed by using the GOR [33, 341 and the Chou-Fasman [35] procedures.

RESULTS CD of bSD in aqueous buffer. The far-ultraviolet CD spectrum of bSD, i.e. the modified basic subdomain peptide of c-Jun, dis- played a weak minimum ( [ H I = - 1200) at 225 nm and a strong minimum ([ 191 =, - 15 000) at 200 nm, in sodiudpotassium phosphate buffer, pH 7, 0.2 mM EDTA, I of 0.1, at both 20°C and 2"C, as shown in Fig. 1. Thus, modifying the temperature of the peptide solution left the spectrum of bSD unchanged. The presence of a strong minimum at 200 nm reflected the large content of unordered structure in bSD. In contrast, the weak minimum at 225 nm could signify the presence of a weak amount of a helix or the existence of a mixture of nascent helix, 3,, helix and a helix [36].

CD of bSD complexed to the CRE DNA site. The binding of bSD to the CRE-containing hexadecanucleotide was demon- strated by the results of CD difference spectroscopy also re- ported in Fig. 1. The CRE containing oligonucleotide alone ex- hibited in sodiudpotassium phosphate buffer, 20"C, a CD spectrum typical for a B-DNA conformation characterized in near-ultraviolet by two strong bands, one positive at approxi- mately 280 nm and one negative at approximately 250 nm; and, in far-ultraviolet by two other positive bands at approximately 225 nm and approximately 190 nm (Fig. 1). Only small varia-

Krebs et al. ( E m J. Biochern. 231) 373

180 190 200 210 220 230 240 250 260 navelength (MI)

Fig.2. Effect of trifluoroethanol on CD spectra of cJun bSD. The spectra correspond to different trifluoroethanol/H,O percentages (vol. trifluoroethanol addedtotal vol.) : 0 % (- -) ; 5 %, (. . . . .) ; 15 %, (-) ; 25%, (-.-); 35%, (--); 50%, (---); 75% (....) and 90%, (-) at 20°C. The insert shows mean residue ellipticity [O] as a function of trifluoroethanol percentage at three wavelengths (191, 208 and 222 nm).

tions were observed in the near-ultraviolet region upon mixing the peptide with DNA, suggesting that the changes affecting the far-ultraviolet spectrum are essentially due to the peptide and only a little due to the oligonucleotide. In agreement, no signifi- cant conformational deviations relative to regular B-DNA have been pointed out in the crystal structure of CRE complexed with Gcn4 [4]. In contrast to DNA, the peptide displays large confor- mational changes during complexation : the CD difference spectra showed a substantial increase of the minima at approxi- mately 208 nm and approximately 222 nm, probably due to the peptide chromophore submitted to a new spatial environment in the complex. The results were consistent with a peptide assum- ing an a-helix stabilization, stronger at 2°C than at 20°C (35% and 20% of a helix, respectively; using the method of Zhong et al. [24]).

CD of bSD in trifluoroethanol/H,O. Trifluoroethanol was then used to follow the propensity of bSD to adopt a helix structure in a more hydrophobic solvent. As illustrated in Fig. 2, addition of trifluoroethanol to the aqueous solution of peptide greatly in- fluenced the shape of dichroic spectra. From 0 to 7 5 % trifluoro- ethanol, the ellipticity of bSD decreased both at approximately 208 nm and approximately 222 nm and increased at approxi- mately 191 nm, while an isodichroic point appeared at approxi- mately 204 nm reflecting a situation dominated by a two-state transition presumably between unordered-to-helix conforma- tions. Most of the spectral variations occurred between 0 and 50 % trifluoroethanol. At higher trifluoroethanol concentrations, i.e. 75 -90 % trifluoroethanol, the spectral variations became in- significant. The main CD results for the secondary structures of bSD are given in Table 1. In these, the unordered structure of a polypeptide is obviously more difficult to define than its coun- terparts, a-helix and &sheet structures. In CD, the term unor- dered could embrace a variety of conformations including ex-

Table 1. Percentages of predicted secondary structures in cJun bSD determined from CD and FTIR. Spectra were recorded at 20°C in aqueous solution and in 90% trifluoroethanol for peptide concentrations of 23 pM and 11.7 mM for CD anf FTIR experiments, respectively.

Tech- Method nique

Proportion of secondary struc- ture type

a-helix strand turn un- ordered

%

CD Yang et al. [22] 0 29 18 53

14 27 54

CD Yang et al. [22] 65 1 6 28

FTIR decomposition 51 27 12 10

Zhong et al. [24] (manual) 4 FTIR decomposition 5

Zhong et al. [24] (manual) 60

l a I

I710 1670 1630 1590 1550

WAVENUMBER (cm-1)

1710 1670 1630 1590 1550 WAVENUMBER (cm-I)

Fig. 3. Analysis of the amide I' region of the FTIR spectra of c-Jun bSD. Spectra were recorded at 20°C in, (A) D,O; (B) trifluoroethanol 90%/D20 10%. The figure contains the experimental absorption spectrum (-), the individual components (-) and their sum (. . ' .). Each component's position was taken from Fourier fourth-derivative and included in the curve-fitting analysis.

tended helix [37] or nascent helix (transient i++ 3 hydrogen bonding) and 3,, helix (i+i+3 hydrogen bonding) [36].

FTIR. The FTIR spectra between 1710 cm-' and 1550 cm-' of the fully H-D exchanged bSD peptide in solution in D,O and trifluoroethanol/D,O (90 %/lo %), after substraction of the re- spective buffer spectra, seemed to have specific features (Fig. 3). Such spectra were constituted of several component bands char- acteristic of specific secondary structures and side-chain contri- butions [38-441. The mathematical methods, fourth-derivative and Fourier self-deconvolution used for band narrowing and res- olution enhancement (data not shown), were applied to detect the underlying structure of the complex amide I' band contour (1700-1620 cm-') [27-29, 401. Both procedures reflected the high complexity of the bSD structure in aqueous and organic solvents and its high sensitivity to medium change. The quantita-

374 Krebs et al. ( E m J. Biochem. 231)

N 1 4 e

K m& - 2 0 ~

24,32 R&& I I I

8.8 8; 6 8.4 8.2 F 2 (ppml

Fig.4. Region of two-dimensional 'H NMR HOHAHA spectrum of c-Jun bSD in aqueous solution. The spectrum was recorded in H,O 90%/ D,O 10% at 500 MHz, 20°C, pH 3.0, 2.1 mM, mixing time 100 ms. Solid vertical lines connect the cross-peaks between the amide and side-chain proton resonances. They further indicate the nature and position in the sequence for all residues, except for the overlapped Lys and Arg resonances (annotated by rectangles).

tive contribution of each band to the total amide I' contour was then evaluated by curve-fitting (see Experimental Procedures). These results are also presented in Fig. 3. The large number of arginine side-chains (9 Arg residues are present in bSD) is re- sponsible for the relative wide and intense peaks at approxi- mately 1584 cm-' and 1609 cm-', while the three Glu 4-car- boxyl groups contribute to the peak at 1562 cm-' [39-451. Con- tribution of these side-chain components was included in the spectral analysis to avoid the approximation otherwise incurred with addition of sloping baseline parameters. Results on the sec- ondary-structure contents for both aqueous and trifluoroethanol solutions were obtained as shown below.

bSD in D,O. The curve-fitting analysis for bSD in D,O is shown in Fig. 3A. The peak frequencies at 1644 cm-' and 1655 cm-' were assigned to unordered or extended helix [37] and a-helix structures, respectively. Those at 1628 cm-' and 1664 cm-' were considered characteristic of P-sheet conforma- tions and turn-like structures, respectively [40, 41, 43, 461. The assignment of the remaining two bands at 1673 cm-' and 1687 cm-' was less obvious since they have been alternatively associated with the high-frequency components of the amide I' vibration of either p sheets I401 or j3 turns 147-491. However, recent FTIR studies realized on model polypeptides and proteins with very high P-sheet or p-turn contents have shown that the contribution of P-sheet structures is negligible between 1670- 1690 cm-' [50-521 while in contrast that of the P turn is always significant around 1675 cm-' [53]. Accordingly, we concluded from the presence of the 1673-cm-' and 1687-cm-' bands in our spectra, the existence of turn-like structures rather than P-sheet structures in bSD. Thus, the FTIR analysis, like CD data, suggested that bSD is practically devoid of stable characteristic a-helix structure in aqueous solution (Table 1) but contains turns and eventually other types of less stable helical struc- tures.

bSD in triJuoroethunol/D,O. The curve-fitting analysis for bSD in trifluoroethanol/D,O (90%/10%) provided a pattern of peaks showing frequencies shifted by about 3 cm-' compared to their counterparts in water (Fig. 3B). The peaks observed at

1628, 1641 and 1651 cm-' were assigned to the sheet, unor- dered, a helix, respectively; and those two at 1666cm-' and 1676 cm-' to P turns. However, the peak frequency found at approximately 1687 cm-' for bSD in water and assigned to P turn structures had disappeared from the spectrum in trifluoro- ethanol. In contrast, the a-helix band was now the strongest and accounted for around 51 % of the total secondary-structure com- ponents (Table 1).

NMR. bSD in H,O. Assays on the behavior of bSD in aqueous solution were made by one-dimensional 'H-NMR for both con- centration and temperature effects. Only small effects were de- tected in bSD spectra between approximately 1.2 mM and 8.6 mM, most of them being confined in the amide region. In addition two-dimensional NMR spectra of peptides in H,O solu- tions at pH 3.0 were recorded at several temperatures between 5°C and 60°C. In the following, we shall concentrate on data obtained at 20°C, since at this temperature, conditions for spectral resolution were found to be optimal. We applied Wiithrich's strategy [54] based on two-dimensional spectra and which consists in the identification of a spin system characteris- tic of each residue, followed by the sequential assignment of adjacent amino acids using the dipolar (NOE) information. Inter- pretation of TOCSY and NOESY spectra was difficult due to the particular composition and sequence of the examined peptide, considering for instance the high proportion of long charged side-chain residues (7 Lys and 9 Arg) which contributed to the large overlap of individual resonances. Yet, spin systems of every amino acid in the sequence, such as Metl2, Asnl4, Va116, Leu33, Tyr34 and Gly35, were immediately assigned in TOCSY spectra (Fig. 4). The aH spectral region appeared particularly narrow, making the detection of several NOE difficult or even impossible. Moreover, the poor structural organization and the apparent flexibility of the peptide in aqueous solution contrib- uted to the difficulty in limiting the number of sequential NHr- NHItl NOES (dNN), even at long mixing-time. In these condi- tions, only several short segments of bSD were assigned through

Krebs et al. ( E m J. Biochem. 231) 375

Serl Gln2

Glu3

Arg4

Ile5

Lys6

Ala7

Glu8

Arg9

LyslO

Argll

Met12

Argl3

Asnl4

Argl5

Val16

Ala17

Alal8

Serl9

Lys20

Ser21

Arg22

Lys23

Arg24

Lys25

Lys26

Glu27

Arg28

PPm ~

* 8.87

8.61

8.5

8.19

8.42

8.28

8.36

8.43

8.36

8.43

8.4

8.44

8.51

8.41

8.25

8.47

8.37

8.25

8.43

8.23

8.39

8.27

8.35

8.43

8.44

8.45

8.52

Table 2. Assignment of c-Jun bSD 'H chemical shifts in H,O. Spectra were recorded in aqueous solution at 20"C, pH 3.0. Chemical shifts are referenced to internal 3-trimethylsilyl-propionate.

Residue Chemical shift of

HN Ha Hp Hy HS HE

4.19 4.4

4.3

4.31

4.09

4.22

4.24

4.28

4.3

4.28

4.3

4.46

4.31

4.67

4.33

4.06

4.33

4.28

4.39

4.27

4.4

4.29

4.24

4.28

4.26

4.27

4.35

4.33

4.01 2.1 1 2

2.07 1.99

1.77 1.82

1.85

1.79 1.76

1.38

2.07 1.99

1.77 1.85

1.79 1.76

1.77 1.85

2.63 2.55

1.76 1.82

2.75 2.81

1.74 1.82

2.04

1.38

1.4

3.9 3.86

1.74 1.79

3.86

1.74 1.79

1.79 1.76

1.71

1.76

1.76 1.8

2.06 1.95

1.76 1.8

2.38

2.44

1.63

0.9 1.2 1.49

1.47 1.43

2.44

1.63 1.67

1.42

1.63 1.67

2.08 2.03

1.63

6.94 7.63

1.6

0.94

1.45 1.38

1.61

1.45 1.4

1.54 1.5

1.45 1.39

1.45 1.39

2.41

1.63 1.56

6.92 :NH2 7.6 :NH2

3.2 NH: 7.23

0.84

1.68 2.99 NH3: 7.56

3.2 NH: 7.23

1.68 3 NMH3: 7.56

3.2 NH: 7.23

CH3: 2.11

3.2 NH: 7.23

: NH2 : NH2

3.16 NH: 7.23

1.67 2.99 NH3: 7.56

3.2 NH: 7.23

1.68 2.98 NH3: 7.56

3.14 NH: 7.12

1.68 3 NH3: 7.56

1.68 3 NH3: 7.56

3.19 NH: 7.23

Table 2. (Continued)

Residue Chemical shift of

HN Ha HP Hy HS HE

PPm

Ile29 8.29 4.14 1.83

Ala30 8.46 4.28 1.37

Lys31 8.27 4.24 1.79 1.76

Arg32 8.35 4.28 1.71

Leu33 8.32 4.27 1.74

Tyr34 8.17 4.62 3.13

Gly35 8.13 3.88 3.85

2.9

0.9 1.18 1.45

1.45 1.4

1.54 1.5

1.49 1.41

0.84

1.68 2.98 NH3: 7.56

3.14 NH: 7.12

0.87 0.81

2,6H: 7.13 3.5H: 6.82

- cJun

daN

INN

daN

dNN

daiNi+3

daiPi+3

- n i 10 20 30 36

Gcn4 VPESSD PA ALK R A RNT EAARRSRA RK L Q R MKQCEDKV

H20 : bSD

: Fragments

: bSD

: Fragments

TFE 50%

: bSD

: Fragments

: bSD

: Fragments

: bSD

: Fragments

: bSD

: Fragments

Fig. 5. Qualitative NOESY connectivity schemes for c-Jun bSD pep- tide and its fragments (NP and CP) in aqueous and trifluoroethanol 50 % solutions. Comparison with Gcn4 bSD in aqueous solution. Ex- periments were performed at 20"C, pH* 3.0 (uncorrected value). For trifluoroethanol 50%, only the i-i+3 connectivities are reported. (*), large signal overlapping. Below is shown the sequential dNN connectivi- ties from Saudek et al. [ l l ] for Gcn4 bSD (residues 220-256) aligned according to c-Jun bSD sequence. Experimental conditions: H,O 90%/ D,O lo%, 37°C pH 3.5,4 mM.

3 76 Krebs et al. ( E m J. Biochern. 231)

0.4

n 0.3

E a 0.2 W

-0.2 U Q I -0.3 V Q u -0.4

I

I

-0.5

-0.6

B . 0.2

i'i 0.1 + I 1

- -0.1 8 -2 5 -0.2 a

I I t Y

-0.4 1 \ I Fig.6. Effect of trifluoroethanol on cJun bSD 'H chemical shifts. (A) Histogram of the calculated differences on N,, (W) and aH (0) chemical shifts for assigned bSD residues between trifluoroethanol SO% and H,O at 20°C, approximately pH* 3.0 (uncorrected value). Amide proton chemical shift temperature coefficients in SO % trifluoroethanol are shown at the top of the N, chemical shift differences. Coefficients higher than approxi- mately -S/-6 ppb/K indicate residues whose amides are hydrogen bonded intramolecularly [5S]. (B) Profile of differences for aH chemical shifts between SO% trifluoroethanol and random coil values (0) or H,O and random coil values (A) at 20"C, approximately pH* 3.0 (uncorrected value). Random coil values were taken from Wuthrich [S4, 621. In the two gaps (Arg9-Argl3 and Serl9-Lys25) of the assignments in SO% trifluoroetha- nol, only the (H,O-random coil) differences are reported.

the sequential doN and dNN NOE. At the end, ambiguities per- sisted for 12 LyslArg residues and their neighboring residues.

To improve the score of proton assignments, we resorted to a two-fragment strategy, the bSD peptide being divided into two almost equal parts, namely the NP peptide (19 residues) and the CP peptide (20 residues), and overlapping over the four central residues Val-Ala-Ala-Ser (positions 16- 19) of bSD (see Experi- mental Procedures). This allowed, in particular, the separation of the two Arg-Lys-Arg repeats, Arg-Lys-Arg (positions 9- 11)

in NP and Arg-Lys-Arg (positions 22-24) in CP. The procedure proved to be particularly useful as it provided the complete as- signment of the bSD 'H chemical shifts for aqueous solution. The values presented in Table 2 demonstrate the complexity of signal distribution in the spectrum of bSD. For instance, the N,, signals of 25 residues, among the 35 residues of bSD are con- fined between 8.47 ppm and 8.27 ppm, i.e. within 0.2 ppm. It was noticeable that comparison of chemical shifts of residues Gln2, Glu3, Ile5 to Glu8, Met12, Asnl4, Va116, Ile29, Ala30,

Krebs et al. ( E m J. Biochem. 231) 377

Leu33 assigned in both the fragments and the bSD peptide re- vealed only weak differences, namely 0.05 ppm for each proton of the total spin system of each residue.

The spectral resemblence between bSD and its constitutive fragments was further confirmed by the NOE connectivity schemes presented in Fig. 5. The sequential dNN NOE were de- tected especially in four separate short segments : Gln2-Ala7, Argl5-Alal7, Lys20-Arg22 and Arg28-Tyr34, well distrib- uted from the N-terminal end to the C-terminal end of bSD. Yet, NOE connectivities as dN,N,+,, da,N,+,, da,N,+,, cia$,+,, da,N,+4, typical of helical structures [54] were neither detected for bSD nor the fragments in aqueous solution. The a,/N, cross-peaks produced by bSD residues Gln2, Glu3, Ile5, Lys6, Metl2, Asnl4, Va116, Ser21, Ile29, Tyr34, Gly35 were sufficiently re- solved in the DQF-COSY spectrum to estimate the apparent coupling constants, 3J,N, useful for conformational analysis. The measured values in bSD were noticeably higher (> 6 Hz) com- pared to the small values (=3-4 Hz) generally found for a heli- ces [54]. Most of these values were confirmed through the analysis of the fragments.

The temperature dependence of the amide proton chemical shifts were considered as suitable for the detection of hydrogen bonds. Temperature coefficient values higher than -5/-6 ppbl K are generally indicative of solvent shielding or hydrogen bonding [55]. All the bSD residues showed very low and rather uniform values around -8 2 1 ppb/K, reflecting the absence of intramolecular hydrogen bonds within the peptide in aqueous solution [55].

bSD in tr@uoroethanol/H,O, Examination of bSD one-di- mensional NMR spectra recorded at increasing concentrations of trifluoroethanol in H,O (0, 15, 25, 30, 35, 50 and 80% vol- ume) showed a global upfield shift of both N, and a, protons. This reflects the stabilization of the overall bSD molecule into helix [56], further confirmed by the degeneracy of the a, reso- nances [57, 581. Unfortunately, because of the viscosity of the solution [59, 601, the line broadening and aggravation of signal overlap in H,O/trifluoroethanol mixtures prevented the complete assignment of bSD residues, especially for those Lys and Arg within the two segments Arg9-Argl3 and Serl9-Lys25. In that case also, fragments were particularly useful for assignment of bSD in trifluoroethanol 50%.

Fig. 6A presents the chemical shift differences for both the N, and a, protons for the 22 residues assigned in both aqueous and trifluoroethanol solvents. The figure includes the N, temper- ature coefficients determined in trifluoroethanol 50%. The up- field shift demonstrated by the N, and a, resonances (negative difference between trifluoroethanol 50 % and H,O) is significant of helix stabilization by trifluoroethanol in bSD, excluding the terminal residues [56, 611. The concomitant appearance of sev- eral new dNN NOE connectivities, better visualized in fragments, is also in good agreement with a a-helix stabilization (Fig. 5). The merging points were as follows.

In the N-terminal part of bSD, the Arg4 and Ile5 residues demonstrated a N, temperature coefficient of approximately - 2 ppb/K in 50% trifluoroethanol solution, suggesting forma- tion of a hydrogen bond for the corresponding amide groups. Typical helical NOE were concomitantly detected in this part of bSD and the corresponding NP peptide, between a, of Gln2 and pH, 7-6 CH, of Ile5 (Fig. 5).

In the central part of bSD, the typical helical NOE arising between the a, of Asnl4 and the Po, of Ala17 (also visible in NP) reflected the organization into helix of this part of the mole- cule. The N, temperature coefficient of the Val16 residue (-6 ppb/K) was further indicative of a weak hydrogen bond in- volving the corresponding amide group, also consistent with.the a-helix structure.

In the C-terminal part of bSD, the largest upfield shifts were observed on the N, of Arg32 and the a, of Ile29. The observa- tion of sequential dm and long-range darb+, (Lys26llle29 and Ile29/Arg32) and d,,,,,, (Ile29/Arg32) cross-peaks in trifluoro- ethanol/H,O further attested to the stabilization of this segment’s residues in a helical structure. Among these, Arg32 further ex- hibited a N, temperature coefficient of approximately - 1 ppb/ K reflecting its implication in a hydrogen bond.

All together, we observed a good correlation between the deviations of the N, chemical shifts induced by trifluoroethanol and the NH temperature coefficients in the same solvent. Particu- larly illustrative were the residues Ile5 and Arg32 which both were found within helical segments and which displayed large N, deviation and very high N, temperature coefficient. How- ever, the low N, temperature coefficients (-13, -13, -16 ppb/ K, respectively) exhibited by Gln2, Leu33 and Tyr34 residues could be explained by their position at the termini of the peptide which prevented their implication within a stable helical struc- ture.

Fig. 6 B shows the a, proton chemical shift deviations from the aqueous solution random values reported by Wiithrich [54, 621 for tetrapeptide residues and our values determined in aque- ous and trifluoroethanol solutions. For bSD in aqueous solution, we observed negative deviations confirming the presence of sev- eral sites of nascent regular structures [II , 15, 581 suggested by the dNN NOE. As expected, trifluoroethanol developed the a, deviations for residues contained in these sites and more weakly for residues located at their borders, thus demonstrating the cou- pled helix-stabilizing and helix-propagating effect of trifluoro- ethanol [63, 571.

DISCUSSION

At the onset of this spectroscopic study, it was not clear whether the 35-residue bSD peptide of c-Jun would be devoid of stable a-helical structure in aqueous solution, as the traditional secondary-structure predictions performed with the procedures of GOR [33, 341 and of Chou-Fasman [35] disclosed a high propensity for bSD to adopt a helical structure along the whole amino acid sequence (data not shown). The similar bSD se- quence of Gcn4 also presented a helical structure profile, al- though the six N-terminal residues and the four C-terminal resi- dues of the sequence were predicted to be unordered. These re- sults are in perfect agreement with the a-helix conformations exhibited by the cJun and Gcn4 bSD peptides in their crystal complex with AP-1 [5] and AP-l/CRE [3, 41, respectively.

Nascent helical structures for bSD in aqueous solution. Our CD and FTIR data established that the various type helices, ex- tended strands or turns constitute 45% of the total secondary structure of bSD in aqueous solution. The NOE patterns were in agreement with the CD and FTIR measurements. They provided a folded content of about SO%, since 19 residues of bSD give rise to NHi-NHI+, NOE in aqueous solution. For NMR experi- ments, the term folded was preferred to helical as it includes structures as different as turns, a and 3,, helices which all elicit NHi-NHi+l NOE [64, 651. Such NOE arose in the four stretches Gln2 -Ala7, Argl5 - Alal7, Lys20-Arg22 and Arg28 -Tyr34, where they were however found weaker than those aHi-NIli+, . Globally, the results from NOE together with those derived from the available ,JNC coupling constants, a, chemical shifts and N, temperature coefficients, confirmed the CD and FTIR findings and further indicated that the above mentioned four stretches assume nascent helical structures or extended helix conforma- tion [36, 37, 581.

378 Krebs et al. (Eur. J. Biochem. 231)

a-helix stabilization at the nascent helix sites in trifluoroetha- no1 solution. The degree of a-helix content of bSD was highly increased in the more hydrophobic medium provided by tri- fluoroethanol. The magnitude of conformational change detected in bSD by both the CD and FTIR analysis is not so common for natural peptides [63, 66-67]. The significant exceptions are several amphiphilic peptides which show CD spectra with maxi- mum helical content already at low trifluoroethanol ratios (30- 50%) [59, 63, 67-71]. Moreover, the CD profiles for elliptici- ties vs trifluoroethanol ratios at either 191, 208 or 222 nm re- flected a weak cooperative process also suggestive of the rela- tive ease for bSD to undergo structural transitions upon change of surrounding. The NMR measurements further indicated that the a-helix stabilization promoted by trifluoroethanol occurs to a large extent within the segments presenting nascent or extended helical structures in aqueous solution. Illustrative effects were the accentuated upfield chemical shifts of the a, resonances and the increased N,, temperature coefficients for residues located in these segments. The concommitant appearance or strengthening of N,,-N,, NOE, with, above all the emergence of long-range NOE, brought further support to the a-helix stabilization affect- ing the nascent helical segments in trifluoroethanol.

The properties shown by bSD were thus consistent with pre- vious reports where peptides with inherent helical propensity, generally predicted by sequence-based algorithms, are further stabilized by trifluoroethanol [63, 65, 72, 731. The parameters of such algorithms [33-351 are however issued from proteins and could reflect the environments created by the tertiaqdquater- nary structure of the protein. Thus, in stabilizing the a-helix con- formation of the bSD peptide fragment, trifluoroethanol should mimic the environments created by the whole cJun protein on the peptide segment corresponding to bSD.

c-dun interacting with CRE. The ability of the c-Jun bSD pep- tide to adopt an a-helix structure during its association with CRE probably could reflect the ease of this peptide to undergo the unordered(inc1uding nascent helix and extended structures)-to- helix transition, revealed in trifluoroethanol. The fact that in CD experiments the stabilization of extra helix structure does not exceed 20% (20°C) or 35% (2"C), for the 2 : l peptide/DNA complex was consistent with an exchange process governed by a relatively weak association constant. A possible reason for that could be inherent to the size of the peptide itself which lacks the leucine-zipper motif and which is thus unable to form di- mers. The dimer formation appears of importance since it is in- volved in the stabilization of the peptideDNA complex [14, 741. Another reason could be the rather high ionic strength ( I = 0.1) which has been used in the present DNA-peptide binding experi- ments. This ionic strength was chosen to avoid the complex pre- cipitation through non-specific ionic interactions but could also result in a weakening of the peptide/DNA complex stability.

Comparison between cJun and Gcn4 basic subdomains. It then seemed interesting to compare our results on cJun with those previously reported for Gcn4 [lo, 111, despite the exis- tence of differences in experimental conditions (pH, temper- ature). CD experiments in aqueous solution provided a lower content of a helix in the bSD peptide of c-Jun (5 %) than that of Gcn4 (1 5 %), and do not really conform to the secondary-struc- ture predictions [33 -351. However, the two peptides exhibited a similar unordered-to-helix transition with a same ratio of a helix (50 %) at 50 % trifluoroethanol. More noticeable differ- ences were reflected by NMR experiments performed in aqueous solution. Where Gcn4 bSD presented an unique and continuous stretch of dNN connectivities along the sequence (residues 7 -23 [I I ] ; Fig. 5), the c-Jun bSD exhibited four short discrete

stretches. Moreover, in the d,,-exhibiting segments, the mea- sured values of the 'JCN coupling constants (G5 Hz in Gcn4 [I l l and 2 6 Hz in c-Jun) reflected a more pronounced helical struc- ture for Gcn4 than c-Jun. However, the differences in aH chemi- cal shifts between c-Jun and Gcn4 (data not shown) were sur- prisingly weak in the central segment of bSD implicated in the DNA binding and containing the important conserved residues (LyslO, Argll, Argl3, Asnl4, Ala 17, Ala 18, Ser21, Arg22, Arg24, Lys25).

Thus, the bSD peptides of c-Jun and Gcn4 display several small differences. However, they seem to be characterized by a similar high conformational flexibility which could be essential for the DNNprotein complex formation.

Conclusion. Joint CD, FTIR and 'H-NMR experiments pro- vided correlated results revealing the absence of clear conforma- tional preferences for the bSD peptide of cJun in aqueous solu- tion. Presumably, the malleability demonstrated by bSD both in varying the trifluoroethanol content in solution and during asso- ciation to DNA, is central to specific binding of the c-Jun pro- tein, and more generally basic zipper proteins to DNA. During the binding process of c-Jun and probably of also Gcn4, peptide flexibility is required to insure the adjustment of interacting groups, especially for the oligonucleotide itself generally dis- plays a quasi-unvarying structure. Such a conclusion has been recently reached by Spolar and Record 1751 during their review- ing of a series of available DNNprotein complex structures, in- cluding CRE-Gcn4 [4].

We gratefully acknowledge support for this study from Z'Association de la Recherche sur le Cancer and La Ligue Nationale Contre le Cancer. D. K. has a fellowship from La Ligue Nationale Contre le Cancer. We thank J. P. Levillain and M. Le Maout for their skilled technical assis- tance in peptide synthesis, Dr E. Lescot for providing oligonucleotides, G. Tevanian for generous help in using NMR equipment and Prof. B. Alpert for access to the FTIR spectrophotometer.

REFERENCES 1. Curran, T. & Vogt, P. K. (1992) in Transcriptional regulation

(McKnight, S. L. & Yamamoto, K. R.: eds) pp. 797-832, Cold Spring Harbor Laboratory Press.

2. Hurst, H. C. (1994) Protein profle vol. 1 , Transcription factors I , hZIP proteins, pp. 143-144, Academic Press, New York.

3. Ellenberger, T. E., Brandl, C. J., Struhl, K. & Harrison, S. C. (1992) The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted a-helices : crystal structure of the protein-DNA complex, Cell 71, 1229 - 1 231.

4. Konig, P. & Richmond, T. J. (1993) The X-Ray structure of the GCN4-bZIP bound to ATFKREB site DNA shows the complex depends on DNA flexibility, J. Mol. Bid. 233, 139-154.

5. Glover, J. N. M. & Harrison, S. C. (1995) Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA, Nature 373, 257-261.

6. Vogt, P. K., Bos, T. J. & Doolittle, R. F. (1987) Homology between the DNA binding domain of the GCN4 regulatory protein of yeast and the carboxyl-terminal region of a protein coded by the onco- gene jun, Proc. Natl Acad. Sci. USA 84, 33316-33319.

7. Struhl, K. (1987) The DNA-binding domain of the Jun oncoprotein and the yeast GCN4 transcriptional activator protein are function- ally homologous, Cell 50, 841 -846.

8. Struhl, K. (1988) The Jun oncoprotein, a vertebrate transcription factor, activates transcription in yeast, Nurure 332, 649-650.

9. Mauffret, O., Hartman, B., Convert, O., Lavery, R. & Fermandjian, S. (1992) The fine structure of two DNA dodecamers containing the CAMP responsive element sequence and its inverse. Nuclear magnetic resonance and molecular simulation studies, J . Mol. Biol. 227, 852-875.

10. Saudek, V., Pastore, A., Castiglione Morelli, M. A., Frank, R., Gausepohl, H., Gibson, T. & Roesch, P. (1990) Solution structure

Krebs et al. (Eur. J. Biochem. 231) 379

of the DNA-binding domain of the yeast transcriptional activator protein GCN4, Protein Eng. 4, 3-10.

11. Saudek, V., Pasley, H. S . , Gibson, T., Gausepohl, H., Frank, R. & Pastore, A. (1991) Solution structure of the basic region from the transcriptional activator GCN4, Biochemistry 30, 1310-1317.

12. Weiss, M. A., Ellenberger, T. E., Wobbe, C. R., Lee, J. P., Harrison, S . C. & Struhl, K. (1990) Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA, Nature 347, 575- 578.

13. Agre, P., Johnson, P. F. & McKnight, S . L. (1989) Cognate DNA binding specificity retained after leucine zipper exchange between GCN4 and CEBP, Science 246,922-925.

14. Talanian, R. V., McKnight, C. J. & Kim, P. S . (1990) Sequence- specific DNA binding by a short peptide dimer, Science 249,

15. Scholtz, J. M. & Baldwin, R. L. (1992) The mechanism of a-helix formation by peptides, Annu. Rev. Biophys. Biomol. Struct. 21, 95- 11 8.

16. Bos, T. J., Monteclaro, F. S . , Mitsonobu, F., Ball, A. R. Jr, Chang, C. H. W., Nishimura, T. & Vogt, P. K. (1990) Efficient trans- formation of chicken embryo fibroblasts by cJun requires struc- tural modifications in coding and noncoding sequences, Genes & Dev. 4, 1677-1687.

17. Chida, K. & Vogt, P. K. (1992) Nuclear translocation of viral Jun but not of cellular Jun is cell cycle dependent, Proc. Natl Acad. Sci. USA 89, 4290-4294.

18. Kim, J., Tzamarias, D., Ellenberger, T., Harrison, S . C. & Struhl, K. (1993) Adaptability at the protein-DNA interface is an important aspect of sequence recognition by bZIP proteins, Proc. Natl Acad. Sci. USA 90, 4513-4517.

19. Montminy, M. R., Sevarino, K. A., Wagner, J. A., Mandel, G. & Goodman, R. H. (1986) Identification of a cyclic-AMP-respon- sive element within the rat somatostatin gene, Proc. Nut1 Acad. Sci. USA 83, 6682-6686.

20. Talanian, R. V., McKnight, J. C., Rutkowski, R. & Kim, P. S . (1992) Minimum length of a sequence-specific DNA binding peptide, Biochemistry 31, 6871-6875.

21. Fasman, G. D. (1976) in Handbook of biochemistry and molecular biology: Nucleic acids, 3rd edn, vol. 1, p. 589, CRC Press, Cleve- land OH.

22. Yang, J. T., Wu, C. S. C. & Martinez, H. M. (1986) Calculation of protein conformation from circular dichroism, Methods Enzymol. 130, 208-269.

23. Woody, R. W. (1985) Thepeptides (Hruby, V., ed.) vol. 7, Academic Press, New York.

24. Zhong, L. & Johnson, C. W. Jr (1992) Environment affects amino acid preference for secondary structure, Proc. Nut2 Acad. Sci. USA

25. Chen, Y.-H., Yang, J. T. & Chang, K. H. (1974) Determination of the helix and form of proteins in aqueous solution by circular dichroism, Biochemistry 13, 3350-3359.

26. Surewicz, W. K. & Mantsch, H. H. (1989) The conformation of dynorphin A(1-13) in aqueous solution as studied by Fourier transform infrared spectroscopy, J. Mol. Struct. 214, 143- 147.

27. Kauppinen, J. K., Moffat, D. J., Mantsch, H. H. & Cameron, D. C. (1981) Fourier self-deconvolution : a method for resolving intrin- sically overlapped bands, Appl. Spectrosc. 35, 271 -276.

28. Moffatt, D. J. & Mantsch, H. H. (1992) Fourier resolution enhance- ment of infrared spectral data, Methods Enzymol. 210, 192-200.

29. Cameron, D. G. & Douglas, J. M. (1987) A generalized approach to derivative spectroscopy, Appl. Spectrosc. 41, 539-544.

30. Marion, D. & Wiithrich, K. (1983) Application of phase sensitive two-dimensional correlated spectroscopy for measurements of 'H- 'H spin-spin coupling constants in proteins, Biochem. Biophys. Res. Commun. 113, 967-974.

31. Griesinger, C., Otting, G., Wiithrich, K. & Emst, R. R. (1988) Clean TOCSY for 'H spin system identification in macromolecules, J. Am. Chem. Soc. 110, 7870-7872.

32. Plateau, P. & GuCron, M. (1982) Exchangeable proton NMR without base-line distorsion, using new strong-pulse sequences, J. Am. Chem. Soc. 104, 7310-7311.

33. Gamier, J., Osguthorpe, D. J. & Robson, B. (1978) Analysis of the accuracy and implications of simple methods for predicting the

769 - 771.

89, 4462 - 4465.

secondary structure of globular proteins, J. Mol. Bid. 120, 97- 120.

34. Biou, V., Gibrat, J.-F., Levin, J., Robson, B. & Gamier, J. (1988) Secondary structure prediction: combination of three different methods, Protein Eng. 2, 185-191.

35. Chou, P. Y. & Fasman, G. D. (1978) Prediction of the secondary structure of proteins from their amino acid sequence, Adv. Enzy- mol. 47, 45-148.

36. Millhauser, G. L. (1995) Views of helical peptides: a proposal for the position of 3,,-helix along the thermodynamic folging path- way, Biochemistry 34, 3873-3877.

37. Anderson, G. J., Haris, P. I., Chapman, D., Romer, J. T., Toth, G. K., Toth, I. & Gibbons, W. A. (1992) Synthesis and spectroscopy of membrane receptor proteins; The y subunit of the IgE receptor, E m J. Biochem. 207,51-54.

38. Miyazawa, T. & Blout, E. R. (1961) The infrared spectra of polypep- tides in various conformations: amide I and I1 bands, J. Am. Chem. Soc. 83, 712-719.

39. Chirgadze, Y. N., Fedorov, 0. V. & Trushina, N. P. (1975) Estima- tion of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water, Biopolymers 14,679- 694.

40. Byler, M. & Susi, H. (1986) Examination of the secondary structure of proteins by deconvolued FTIR spectra, Biopolymers 25, 469 - 487.

41. Surewicz, W. K. & Mantsch, H. H. (1988) New insight into protein secondary structure from resolution-enhanced infrared spectra, Biochim. Biophys. Acta 952, 115 - 130.

42. Surewicz, W. K., Mantsch, H. H. & Chapman, D. (1993) Determina- tion of protein secondary structure by Fourier transform infrared spectroscopy, Biochemistry 32, 389-394.

43. Krimm, S . & Bandekar, J. (1986) Vibrational spectroscopy and con- formation of peptides, polypeptides, and proteins, Adv. Protein Chem. 38, 181-364.

44. Holloway, P. W. & Mantsch, H. H. (1989) Structure of cytochrome b5 in solution by Fourier-transform infrared spectroscopy, Bio- chemistry 28, 931 -935.

45. Venyaminov, S . Y. & Kalnin, N. N. (1990) Quantitative IR spectro- photometry of peptide compounds in water (H,O) solutions. I. Spectral parameters of amino acid residue absorption bands, Bio- polymers 30, 1243-1257.

46. Prestelski, S . J., Byler, D. M. & Liebman, M. N. (1991) Comparison of various molecular forms of bovine trypsin: correlation of infra- red spectra with X-ray crystal structures, Biochemistry 30, 133- 143.

47. Haris, P. I., Lee, D. C. & Chapman, D. (1986) A Fourier transform infrared investigation of the structural differences between ribo- nuclease A and ribonuclease S, Biochim. Biophys. Acta 874,

48. Jackson, M., Haris, P. I. & Chapman, D. (1989) Conformational transitions in poly(l4ysine) : studies using Fourier transform infra- red spectroscopy, Biochim. Biophys. Acta 998, 75-79.

49. Dong, A., Huang, P. & Caughey, W. S . (1990) Protein secondary structures in water from second-derivative amide I infrared spectra, Biochemistry 29, 3303 -3308.

50. Arrondo, J. L. P., Young, N. M. & Mantsch, H. H. (1988) The solu- tion structure of concanavalin A probed by FTIR spectroscopy, Biochim. Biophys. Acta 952, 261-268.

51. Dousseau, F. & Pkzolet, M. (1990) Determination of the secondary structure content of proteins in aqueous solutions from their amide I and amide I1 infrared bands. Comparison between classical and partial least-squares methods, Biochemistry 29,

52. Kalnin, M. N., Baikalov, I. A. & Venyaminov, S . Y. (1990) Quantitd- tive IR spectrophotometry of peptide compounds in water (H,O) solutions. 111. Estimation of the protein secondary structure, Bio- polymers 30, 1273-1280.

53. Pezolet, M., Bonenfant, S . , Dousseau, F. & Popineau, Y. (1992) Conformation of wheat gluten proteins. Comparison between functional and solution states as determined by infrared spectros- copy, FEBS Lett. 299, 247-250.

54. Wiithrich, K. (1986) NMR ofproteins and nucleic acids, Wiley, New York.

,

255 -265.

8771 -8779.

380 Krebs et al. (Eul: J. Biochem. 231)

55. Dyson, H. J., Rance, M., Houghten, R. A,, Lerner, R. A. & Wright, P. E. (1988) Folding of immunogenic peptide fragments of pro- teins in water solutions: sequence requirements for the formation of a reverse turn, J. Mol. Biol. 201, 161-200.

56. Wishart, D. S., Sykes, B. D. & Richards, F. M. (1991) Relationship between nuclear magnetic resonance chemical shift and protein secondary structure, J. Mol. Biol. 222, 311-333.

57. Storrs, R. W., Truckses, D. & Wemmer, D. E. (1992) Helix propaga- tion in trifluoroethanol solutions, Biopolyrners 32, 1695 - 1702.

58. Dyson, H. J., Rance, M., Houghten, R. A,, Wright, P. E. & Lemer, R. A. (1988) Folding of immunogenic peptide fragments of pro- teins in water solutions: the nascent helix, J. Mol. Biol. 201,201 - 217.

59. Mierke, D. F., Dun; H., Kessler, H. & Jung, G. (1992) Neuropeptide Y. Optimized solid-phase synthesis and conformational analysis in trifluoroethanol, Eul: J . Biochem. 206, 39-48.

60. Bruch, M. D., McKnight, C. J . & Gierasch, L. M. (1989) Helix formation and stability in a signal sequence, Biochemistry 28, 8554-8561.

61. Wishart, D. S., Sykes, B. D. & Richards, F. M. (1992) The chemical shift index: a fast and simple method for the assignment of pro- tein secondary structure through NMR spectroscopy, Biochemis- try 31, 1647-1651.

62. Bundi, A. & Wuthrich, K. (1979) 'H-NMR parameters of the com- mon amino acid residues measured in aqueous solutions of the linear tetrapeptides H-Gly-Gly-X-L-Ala-OH, Biopolymers 18, 285 -297.

63. Jimenez, M. A,, Bruix, M., Gonzalez, C., Blanco, F. J., Nieto, J. L., Herranz, J. & Rico, M. (1993) CD and 'H-NMR studies on the conformational properties of peptide fragments from the C-termi- nal domain of thermolysin, Eul: J. Biochem. 211, 569-581.

64. Bradley, E. K., Thomason, J. F., Cohen, F. E., Kosen, P. A. & Kuntz, I. D. (1990) Studies of synthetic helical peptides using circular dichroism and nuclear magnetic resonance, J. Mol. Biol. 215, 607-622.

65. McLeish, M. J., Nielsen, K. J., Najbar, L. V., Wade, J. D., Lin, F., Doughty, M. B. & Craik, D. J. (1994) Conformation of a peptide corresponding to T4 lysozyme residues 59-81 by NMR and CD spectroscopies, Biochemistry 33, 11 174- 11 183.

66. Greff, D., Toma, F., Fermandjian, S., Low, M. & Kisfoludy, L. (1976) Conformational studies of corticotropin 1-32 and consti- tutive peptides by circular dichroisnl, Biochim. Biophys. Acta 439, 219-231.

67. Sonnichsen, F. D., Van Eyk, J. E., Hodges, R. S. & Sykes, B. D. (1992) Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide, Biochemis- try 31, 8790-8798.

68. Lau, S. Y. M., Taneja, A. K. & Hodges, R. S. (1984) Effects of high-performance liquid chromatographic solvents and hydropho- bic matrices on the secondary and quaternary structure of a model protein. Reverse-phase and size-exclusion HPLC, J. Chromatogl:

69. Nelson, J. W. & Kellenbach, N. R. (1989) Persistence of the a- helix stop signal in the S-peptide in trifluoroethanol solutions, Biochemistry 28,5256-5261.

70. Lehrman, S. R., Tuls, J. L. & Lund, M. (1990) Peptide u-helicity in aqueous trifluoroethanol : correlations with predicted a-helicity and the secondary structure of the corresponding regions of bo- vine growth hormone, Biochemistv 29, 5590-5596.

71. Zhou, N. E., Mant, C. T. & Hodges, R. S. (1990) Effect of preferred binding domains on peptide retention behavior in reversed-phase chromatography : amphipathic a-helices, Peptide Res. 3, 8 -20.

72. Sanford, D. G., Kanagy, C., Sudmeier, J. L., Furie, B. C., Furie, B. & Bachovchin, W. W. (1991) Structure of the propeptide of prothrombin containing the y-carboxylation recognition site deter- mined by two-dimensional NMR spectroscopy, Biochemistry 30, 9835-9841.

73. Willhold, D., KrLiger, U., Frank, R., Rosin-Arhesfeld, R., Gazit, A,, Yaniv, A. & Rosch, P. (1993) Sequence-specific resonance assign- ments of the 'H-NMR spectra of a synthetic, biologically active EIAV Tat protein, Biochemistry 32, 8439-8445.

74. Cohen, D. R. & Curran, T. (1990) Analysis of dimerization and DNA binding functions in Fos and Jun by domain-swapping: in- volvement of residues outside the leucine zipperhasic region, Oncogene 5, 929-939.

75. Spolar, R. S. & Record, M. T. Jr (1994) Coupling of local folding to site-specific binding of proteins to DNA, Science 263, 777 - 784.

317, 129-140.