Eur. J. Biochem. 187,645-650 (1990) FEBS 1990

c>

NMR identification of a partial helical conformation for bombesin in solution John A. CARVER' and J . Grant COLLINS' Department of Chemistry, The University of Wollongong, Australia Department of Chemistry, University College, University of New South Wales, Canberra, Australia (Received July 25/October 18, 1989) - EJB 89 0922

The conformation of bombesin in trifluoroethanol/water mixtures has been studied using H-NMR spectroscopy. By a combination of two-dimensional 'H-NMR techniques and measurement of vicinal NH-U-CHspinspin coupling constants, the secondary structure of the molecule has been determined. Bombesin adopts a helical structure in the region from Asn-6 to Met-I4 with the remaining N-terminal portion existing as a more extended structure. The structure is very similar to that proposed from Fourier-transform infrared spectroscopic measurements for bombesin inserted into lipid bilayers [D. Erne & R. Schwyzer (1987) Biochemistry 26, 6316-63191. The absence of a hydrogen bond between the sidechains of Trp-8 and His-12 is discussed in terms of the ionization state of His-12. Stabilisation of the helix results when His-12 is in the ionized state. There has been much research interest of late in the tetradecapeptide bombesin since the discovery that bombesin and related peptides act as potent growth factors for human small-cell lung-carcinoma cell lines [I -31. It has been postulated that these peptides may perform a regulatory role in cell growth [3,4]. In addition, bombesin has a wide range ofpotent pharmacological effects [5]. Bombesin was first isolated from frog skin [6] and found to have the amino acid sequence: Glp-

presented herein are a characterisation by high-resolution 'HNM R of the conformation of bombesin in trifluoroethanol/ water solvent mixtures. Trifluoroethanol mimics membrane environments by encouraging intra-molecular hydrogenbonding interactions and thereby favours ordered structures. The conformation in this solvent mixture is shown to be very similar to that proposed for bombesin in a lipid environment from Fourier-transform infrared studies [I 21.

Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-MetNH2, where Glp is 5-oxoproline (pyroglutamic acid). The mammalian counterparts include the 27-amino-acid gastrin-releasing peptide, neuromedin B and C which have been isolated from the central nervous system, lung and gut [4]. All of the peptides in the bornbesin family share a highly similar C-terminal region, particularly in the final ten amino acids; it has been shown that pharmacological activity is exerted fully by the C-terminal nonapeptide [7, 81. Several structural studies have recently been reported on bombesin and gastrin-releasing peptide. Both peptides, from NMR studies, have been shown to adopt little ordered structure in aqueous solvent or dimethyl sulphoxide [9 - 1I]. However, as determined from Fourier-transform infrared [12], circular dichroism and fluorescence studies [13], when bombesin is incorporated into lipid environments the molecule adopts a partial helical structure. It has been postulated in these studies that the C-terminal region of the molecule adopts a helical structure whilst inserted into the hydrophobic lipid environment, whereas the rest of the molecule is in the aqueous phase and exists in a much less ordered structure. The results Correspondence to J. A. Carver, Department of Chemistry, The University of Wollongong, P.O. Box 1144, Wollongong, NSW 2500, Australia Abbreviations. COSY, correlation spectroscopy; DQF COSY, double-quantum-filtered correlation spectroscopy; RELAY, relayed coherence transfer spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; ' J H N avicinal , NH-K-CHspin-spin coupling constant.

MATERIALS AND METHODS Bombesin was purchased from Auspep Pty. Ltd (Victoria, Australia) and used without further purification. For the 2D-NMR experiments, bombesin was dissolved in either 70% (2H3)trifluoroethanol/30Y~D 2 0 or 70% (2H3)trifluoroethano1/30% HzO lo a concentration of approximately 8 mM and a pH meter reading (pH*) of 3.1. 'H-NMR spectra (COSY, DQF COSY, RELAY and NOESY) were recorded at 300, 400 and 500 MHz on Bruker CXP-300, Jeol GX-400 and Varian VXR-500 spectrometers, respectively, with the mixing time in the NOESY experiment being 250 ms. The NOESY experiment was recorded at 25 "C while all remaining spectra were acquired at 30'C. Spectra on the Bruker and Varian spectrometers were recorded in the phase-sensitive mode using the time-proportional phase-incrementation method [I41 and the method of States et al. [I51 respectively. Spectra recorded on the Jeol GX-400 spectrometer were recorded with P-type peak selection and processed with an absolute value. RESULTS When bombesin dissolved in D 2 0 is titrated with trifluoroethanol, a number of well resolved resonances (assigned in [9]) exhibit distinct chemical shift changes (Fig.1). Most noticeably, the H-2 resonance of His-12 moves upfield by % 0.7 ppm in going from 0 to 60% trifluoroethanol solution. The His-12 H-4 resonance, however, is unaffected. Smaller

646

0

*

IC 7.5

2

-

I

-

...

* .

2

W 8 H-4

W8 H - 7 W8 H-2

HlZ H - 4 W8 H-6 W8 H - 5

'.O

d0

8,'s

ppm

F2

i

Fig. 2.300-MHz C'OSYspectrum qfhomhesin showing the N H to PCH region qf'the spectrum with the assignments indicated

T

M14 E-CH3 2.0

1.5

i

3

A 9 O-CH3

A

T

I

I

20

,

I

40

60

'/TFE

Fig. 1. Chemical .shiJi variation at 400 M H z of selected hombesin resonances as a function of tr@uoroethanol concentration. The initial pH* reading of the D 2 0 solution was 4.3. TFE = trifluoroethanol

shifts are observed for Trp-8 H-5, H-6, H-7; Ah-9 fl-CH3and one of the Val-10 y-CH3 resonances and no solvent-dependent shifts are present for these resonances above 40% trifluoroethanol. Thus, structural changes occur in going from the random coil conformation in water to trifluoroethanol. A solvent ratio of 70% trifluoroethanol/30% aqueous (DzO or HzO) solvent was chosen for all subsequent 2D-NMR studies. The aim of the 2D-NMR studies was to assign the spectrum and hence determine the secondary structure of bombesin in this solvent mixture. The assignment of the 'H-NMR spectrum of bombesin in trifluoroethanol/H,O was undertaken using the sequential method of Wiithrich 1161. The method relies on the observation of NOES from backbone NH protons to the N H proton (NHi-NHi+ and the a-CH proton (ai-NHi+ I ) of the preceding residue in the sequence. Thus, spectra are recorded in protonated solvent after the amino acid spin types have been determined from correlation experiments such as COSY and

RELAY. The NH to a-CH region of a COSY spectrum of bombesin trifluoroethanol/H20 at 30 "C (Fig. 2) shows that 12 of the expected 14 cross-peaks are observed. The weak cross-peak for Glp-1 was present at lower contour levels, while that for Asn-6 was absent due to solvent saturation, but was observed for a COSY spectrum recorded at 45°C. The a-CH region of the spectrum has its resonances concentrated into a small region and RELAY spectroscopy was used to correlate NH resonances with [j-CH resonances and hence sort out ambiguities in spectral assignment in regions of overlap. NH to 0-CH relayed cross-peaks were observed for 11 of the 12 expected amino acid spin systems (Fig. 3) with that for Glp-1 being absent due to a very weak N H to a-CH cross-peak. The concentration of a-CH resonances into a small chemical shift range meant that the a-CH to p-CH cross-peaks also occurred in a crowded spectral region which complicated assignment. The presence of a-CH to 7-CH relayed crosspeaks for Glp-I, Gln-2, Gln-7, Val-10 and Leu-13 facilitated the assignment of these spin systems (data not shown). From the correlation experiments, assignments were obtained for the unique spin systems of Ala-9 and Val-10. For the remaining spin systems, sequence-specific assignments were made from NOESY spectra following categorization of correlated amino acid resonances into types of spin systems [17]. From the pattern of NOEs originating from N H protons, it is possible to draw conclusions as to the secondary structural motifs that are present in the molecule [18]. The assignments are detailed in Table 1. The NH region of the NOESY spectrum of bombesin is shown in Fig. 4 from which it can be seen that a series of NHiNHi + NOEs are observed, starting from Arg-3 and going along the polypeptide backbone to Met-14. No NHi-Ni + NOEs are observed in the segment from Glp-1 to Arg-3. Asn-6 and Trp-8 N H resonances are coincident and the presence of a very strong NOE between Gln-7 and Trp-8 indicates that an NOE also exists between Asn-6 and Gln-7 NH resonances. It is noted that the NHi-NHi+ NOEs in the region from

,

647

P

0 0

8.0 -

F2

8.5 ppm

I

I

!

4.0

,

I

2.0

3.0

porn

F1

Fig. 3. 400-MHz RELAY spectrum of hoinhesin showing both the direct N H to rs-CH cross-peaks and the relayed NH to fl-CH cross-peaks (the latter cr(is.s-peak.s are labelled)

Table 1. Chemical shifts of hornbesin in 70% tri@~oroethanol/30%H 2 0 pH* 3.1, 3 0 ° C Residue

6 for proton NH

a-CH

fl-CH

others

Glp- 1

7.62

4.31

2.11,2.57

y-ckrZ

2.43

Gln-2

8.28

4.31

2.10

y-c€r2

2.42"

Arg-3

8.23

4.35

1.66, 1.79

y-CHZ 6-CHz E-NH

1.68 3.20 7.12

Leu-4

7.88

4.40

1.67

y-CH 6-CH3

1.72 0.87

Gly-5

8.10

4.01

Asn-6

8.09

4.72

2.87

y-NH2

6.55, 7.27

Gln-7

8.57

4.08

2.12

yCH2

2.31"

Trp-8

8.06

4.46

3.38, 3.44

H-2 H-4 H-5 H-6 H-7 NH

1.22 7.57 7.1 1 7.16 7.36 9.63

yCH3

0.97 (pro-R) 1.06 (pro-S)

Ala-9

7.97

4.10

1.46

Val- 10

7.86

3.14

2.06

Gly-11

8.13

3.74

His-I 2

7.78

4.43

2.93, 3.20

H-2 H-4

7.58 7.14

Leu-13

8.03

4.29

1.66, 1.81

y-CH 6-CH3

1.74 0.92

Met-I4

7.91

4.43

2.00, 2.10

Y-CI-I~ 2.60" &-CF13 2.09

' The &NH2 protons of Gln-2 and Gln-7 and the -CO-NHZ protons of Met-I4 were observed at 6.51, 7.23 ; 6.51, 7.08 and 6.67, 7.08 ppm but were not individually assigned.

,

'

'

'

'

I

8.0

8.5

.

/

'

ppm

F1

Fig. 4. 500-MHz N O E S Y spectrum with a mixing time of250 rns of hornhesin slzowin~the NHi to NHi connectivities

Arg-3 to Gly-5 are much weaker than NHi-NHi+ NOEs in the remainder of the molecule. Fig. 5 shows the NH to a-CH region of the NOESY spectrum with NOEs indicated. In particular, for C-terminal acids (Gln-7 onwards) a number of ai-NHi+ NOEs are observed (e.g. Ala-9 a-CH to His-I2 NH and Trp-8 x-CH to Gly-11 NH) which are not present for N-terminal amino acids. In this region, strong ai-NHi+ cross-peaks are present compared to the weaker ai-NHi + I cross-peaks for the Cterminal segment. All ai-NHi+ NOEs were consistent with assignments from correlation spectra. The observed ai-NHi+ NOEs, in addition to those present between cli and and !xi and N H i + 4 resonances, are summarized in Fig. 6. The ai-pi + and ai-NHi+ NOEs were very weak or absent. In

648 addition, a strong NOE between Gln-7 a-CH and the downfield Val-10 y C H 3 resonance was observed. NOESY spectra of a-helical regions of proteins are characterised by a series of strong NHi-NHi + NOEs in addition to ai-NHi+ 3 , ai-NHi+ and ai-fli+ NOEs with relatively weak ai-NHi+ NOEs being present [17, IS]. The 310-helix has a very similar pattern of NOEs except that ai-NHi+ and clipi + NOEs are absent or are relatively weak [19]. The observation of NOEs of the type NHi-NHi + and ai-NHi+ over

Fig. 5. The N U to a-CH region qf'u 500-MHz NOESY spectrum of homhesin in with u mixing time of250 ms. Intra-residue xi-NHi, and inter-residue ai-NHi+ ,,xi-NHi + and ai-NHi + NOEs are indicated. The two unlabclled NOES at the top of the plot are from Trp-8 H-4 at F2 = 7.57 ppm to Trp-8 a-CH(F, = 4.46 ppm) and Ala-9 a-CH ( F , = 4.10ppm)

the length of the polypeptide chain from Arg-3 to Leu-14, with particularly strong NOEs of these types for the region Asn-6 to Met-14, suggests that this region adopts a generally helical conformation that is tighter in the C-terminal portion. The absence of rather strong ri-pi+ and ai-NHi + NOEs suggests that this region may be in a 310- rather than an ahelical conformation [19]. An NOE between Gln-7 a-CH and a Val-10 y-CH3 is also consistent with a helical structure [20]. The values of the vicinal NH-a-CH spin-spin coupling constants (3JHN,) measured from one-dimensional spectra, are in agreement with this postulate. Using the Karplus equation derived from experimental data on polypeptides, the 3 J H N a value for a regular a-helix is 3.9 Hz and for a 310 helix is 4.2 Hz [17]. The observation of a series of 3 J H N a values less than 6 Hz is a good indication of a helical region [21]. From Asn-6 to Gly-11, the ' J I $ N a values are less than 6.0 Hz with greater values outside this region (Fig. 6). It therefore seems that bombesin adopts a tight helical structure in the region from Asn-6 to Gly-11. Prior to this portion of the molecule (Arg-3 to Gly-5), where weaker NHi-Ni + and stronger aiNHi + NOEs are present and 3JHNavalues are around 7 Hz, the molecule is much more flexible, forming a frayed helix suggesting a conforinational equilibrium, that is fast on the NMR timescale, exists between a helical and random coil or extended structure. The first two residues of the molecule (Glp-1 and Gln-2) exist in a totally extended flexible conformation as judged by the absence of any diagnostic NOEs (apart from a strong ai-NHi+ NOE between Gln-2 and Glp-I) and an intermediate 3J11No, value for Gln-2. The Cterminal region from His-I2 to Met-I4 has a succession of NOEs characteristic of a helical region but the 3 J H N a values are greater than 6 Hz suggesting that the helix may be less regular than in the region from Asn-6 to Gly-I 1. Stereospecific assignment of the two y-CH3 groups of Val-10 was possible from the observation of a large (10-Hz) coupling constant between the a-CH and P-CH protons ('JaD. measured from the DQF COSY spectrum) and a strong NOE from Val-10 N H to one of the y-CH3 and a much weaker NOE to the other y C H 3 . The a-CH and B-CH protons are therefore in the trum configuration with the y2-CH3 nearest the N H proton (and hence the strongest NOE) being in the

Fig. 6. A summury of interresidue NOEs involving the N H , a-CH and j-CHpvotons and the 3JHN, vuluc~sforeuch residue. The height of the bars indicates the intcnsities of the NOEs. Thc a i + j i+ NOEs were both very weak. The ' J H N avalues were determined from ID spectra at various tcmpcratures. Those values markcd with a n astcrisk could not be determined at any tempcraturc due to overlap

649

p r o 3 configuration and the y1-CH3 being in the pro-R configuration [22, 231. Furthermore, a similarly sized NOE is observed to each y-CH3 from the a-CH which is consistent with a trans configuration [22]. This conformation is typical for valine residues in helical regions [22]. In addition to the NOEs summarised in Fig. 6 which enabled conclusions to be drawn as to the- conformation of the molecule, a number of NOEs were observed from the Trp-8 aromatic protons. Trp-8 H-4 exhibits NOEs to Ala-9 aCH (Fig. 5) and ,6-CH3 which would explain the downfield shift of Ala-9 p-CH3 resonance (Fig. 1). Trp-8 H-2 also has a weak NOE to Ala-9 B-CH3 while Trp-8 a-NH and H-5 both show an NOE to Gln-7 y-CH,. Furthermore, there were no NOEs present between residues distant by more than four amino acids in the sequence. Consequently, bombesin adopts a linear structure with no folding of the polypeptide chain back onto itself. No NOEs were observed between His-12 H-2 and Trp-8 aromatic protons, although an NOE was present between His-12 a-CH and Trp-8 B-CH2. Due to ring-current-shielding effects of Trp-8, interaction between these two aromatic rings could cause the large upfield shift observed for His-12 H-2 resonance in trifluoroethanol/H,O. In a helix, the two sidechains, being four residues apart, could readily be oriented near each other and, indeed, the presence of an NOE between His-12 a-CH and Trp-8 j-CH2 suggests that this is so. The absence of an NOE between His-12 H-2 and any Trp-8 aromatic protons could be explained by conformational averaging [17] due to the flipping motion and/or movement between various conformational states of the imidazole sidechain. His-1 2 being outside the tight helical portion of the molecule is consistent with the possibility of it being in conformational equilibrium. The chemical shift behaviour with temperature of the aromatic resonances is of interest with His-I2 H-2 resonance having a large temperature-dependent shift (db/dT = - 0.014 ppm/K, i.e. a downfield shift with increasing temperature) while Trp-8 H-7 has an almost negligible temperature dependence in the same direction (db/dT = - 8 x ppm/K). All other aromatic resonances have a small temperature dependence but shift upfield with increasing temperature (db/ d T z 0.005 ppm/K). Thus, increasing temperature causes His-12 H-2 resonance to shift towards its random-coil chemical shift position, suggesting that the interaction between His-I 2 and Trp-8 aromatic rings is not as intimate at higher temperatures, i.e. higher temperatures shift the equilibrium towards a conformation in which the His-12 sidechain is further away from Trp-8. In contrast, His-12 H-2 resonance in D 2 0 has a much smaller temperature dependence over the temperature range 5-70°C ( - 8 . 4 ~l o p 4 ppm/K). There is therefore no indication in aqueous solvent of partial helix formation at low temperature which would be observed from a dramatic upfield shift of His- 12 H-2 resonance. Interestingly, His-12 a-NH resonance in trifluoroethanol/ H 2 0 exhibits no temperature dependence, unlike all other aNH resonances which show db/dT values of = 0.006 ppm/K. This behaviour could be due to the downfield shift at higher temperatures associated with the removal of Trp-8 aromatic ring being balanced by the upfield shift associated with greater exposure to hydrogen-bonding solvent (H20)molecules. The solvent titration data of Fig. 1 are also indicative of a conformational equilibrium existing for His-12 since at intermediate concentrations of trifluoroethanol (30 - 50%) significant broadening of His-12 H-2 resonance was observed, i.e. a conformational equilibrium exists which is intermediate on the

NMR timescale. The absence of a hydrogen bond between Trp-8 and His-12 sidechains (see below) would enhance such conformational flexibility.

DISCUSSION From careful analysis of the NOESY spectrum and 3 J H N a values (Fig. 6), it was deduced that bombesin adopts a mainly helical conformation in trifluoroethanol/H20 solvent, particularly in the C-terminal region. Thus, the N-terminal two amino acids (Glp-I and Gln-2) exist in an extended conformation. The region from Arg-3 to Gly-5 also adopts a flexible conformation that is possibly more structured than the first two amino acids as shown from the presence of a series of weak NHi-NHi + NOEs. The remainder of the molecule from Asn-6 to Met-14 exists as a helical entity (possibly as a 310 helix) which may be slightly frayed over the last three amino acids (His-12 to Met-14). The C-terminal nonapeptide (Asn-6 to Met-14) is fully active [7, 81, which suggests that the formation of a helix in this region may have functional significance. Indeed, this structure of bombesin is very similar to that proposed by Erne and Schwyzer [I 21 from their Fourier transform infrared studies and thermodynamic calculations on bombesin in trifluoroethanol and when intercalated into phospholipid bilayer membranes. They suggest that the region from Gly-5 to Met-I4 adopts a helical conformation which is inserted into the membrane perpendicularly to the plane of the membrane while the N-terminal segment (Glp-1 to Leu-4) exists as an extended coil. Cavatorta et al. [13] have postulated a similar conformation as a result of their circular dichroism and fluorescence studies on bombesin interacting with lipids. Thus, the structure of bombesin in trifluoroethanol/H,O determined from NMR studies is very similar to that in a membrane environment. Trifluoroethanol is a membrane-mimicking solvent in that it encourages intra-molecular hydrogen bonding rather than those with solvent. Similar conclusions have been drawn about the use of organic solvents to mimic membrane environments from NMR studies in trifluoroethanol/H,O on glucagon-type peptides [24] and in methanol on the membrane-spanning peptides, alamethicin [23], melittin [25] and b-hemolysin [20]. When interacting with a membrane and forming the C-terminal helix, Erne and Schwyzer have suggested that Trp-8 and His-12 are involved in a hydrogen bond between the indole N H and the deprotonated imidazole sidechains, respectively [12]. As has been discussed above, an interaction between the residues is ascertained from the presence of an NOE between Trp-8 p-CH2 and His-12 a-CH. A hydrogen bond would cause the lowering of the pK, value of His-12. The pK, values of Lhistidine in 70% trifluoroethanol/30% D 2 0 and D 2 0 are = 5.5 and = 6.0 respectively (unpublished results) and the pK, of His-12 in aqueous solution is z 6.5 [13]. In a pH* titration of bombesin in 70% trifluoroethanol/30% D 2 0 , the His-I2 H-2 resonance showed no chemical shift change from pH* 1 to 4.5, while above this value, as His-12 began to titrate, the spectrum broadened out (data not shown). It was therefore not possible to obtain a pK, value for His-12. The result does, however, indicate that the upfield chemical shift observed for His-12 H-2 (Fig. 1) is not due to the imidazole sidechain being in the deprotonated form and that, at the pH* value (3.1) for which the 2D-NMR studies were undertaken, the imidazole sidechain was protonated. This would argue against there being a hydrogen bond between Trp-8 and His-12 sidechains (in this solvent system at pH* 3.1) and therefore the upfield

650 chemical shift of His 12 H-2 is due to its close proximity to the ring current of the indole ring of Trp-8. The broadening of the spectrum with increasing pH* 5 arise from an alteration in the structure above ~ 4 . must and/or aggregation of the molecule as His-12 ionizes. Indeed, the spectra at higher pH* values indicate that a number of conformations of the molecule exist that are slow or intermediate on the NMR timescale. It is well known that charged groups play a crucial role in stabilizing helices in isolated peptides and proteins [26]. One mechanism involves an interaction between the charge and the helix dipole [26]. Hence, positively charged residues are statistically found at higher frequencies at the C-terminal of helices, while negatively charged residues are found at the N-terminal [27]. Thus, the presence of the charged form of His-12 at the C-terminal of the molecule would stabilize the helix and, with its ionization, the stabilization would be removed and disruption of the helix would result. The interaction between Trp-8 and protonated His-12 may also be enhanced by partial stacking of the two sidechains as has been suggested for the Phe-8/His-12 pair in the ribonuclease S-peptide [28]. The interaction between these two residues is currently under further investigation by determination of the structure of bombesin using distance geometry and energy minimisation techniques [17, 20, 251. Recently, Richardson and Richardson [29] have undertaken a statistical survey of end-group amino acid preferences in helical regions. They found that Asn has a strong preference for the N-terminal commencement of helices while Gly is the dominant amino acid at the C-terminal. Glycine is well known as a ‘helix breaker’ [30]. The results from this NMR study are consistent with these studies in that the highly helical portion of the molecule commences at Asn-6 and is interrupted at Gly-11 where afterwards the helix is not as rigid. The bombesin receptor is believed to be membrane-bound with a protein component of molecular mass 75-85 kDa [4]. The helical conformation of the bombesin molecule in a membrane environment is not necessarily the conformation it adopts when bound to the receptor. Instead, receptor binding may primarily involve membrane binding in a helical conformation prior to a second conformational change, possibly to a P-turn-type structure involving the Val-I0 to Leu-1 3 tetrapeptide [ 3 1331. ~ The conformational flexibility of the bombesin molecule would allow it to adopt such conformation variability [9]. Indeed, Fourier-transform infrared and circular dichroism studies on signal peptides when interacting with membranes show a transition from random coil to p-turn and then helical structures as the peptide goes from aqueous environment to binding to, and subsequently intercalating into, the membrane [34]. We thank Assoc. Prof. Ray Norton for access to NMR facilities, Andrea Hounslow for help with the NMR experiments and Drs John Wallace and John Ballard for their interest and helpful discussions.

REFERENCES 1. Cuttita, F., Carney, D. N., Mulshine, J., Moody, T. W.. Fedorko. J., Fischler, A. & Minna, J. D. (1985) Nature 316, 823-826.

2. Moody, T. W., Bertness, V. & Carney, D. N. (1983) Peptides 4, 683 - 686. 3. Moody, T. W., Carney, D. N., Cuttita, F., Quattrocchi, K . & Minna, J. D. (1985) Life Sci. 37, 105-113. 4. Zachary, I., Woll, P. J. & Rozengurt, E. (1987) Dev. Biol. 124, 295 - 308. 5. Erspamer, V. & Melchiorri, P. (1983) in Neuroendocrine perspecriws, vol. 2 (Muller, E. E. & MacLeod, R. M., eds) pp. 37106, Elsevier, New York. 6. Anastasi, A., Erspamer, V. & Bucci, M. (1971) Exparientia 27, 166- 167. 7. Broccardo, M., Falonieri Erspamer, G., Melchiorri, P.. Negri, L. & De Castiglione, R. (1975) Br. J . Pharmacol. 5.5.221 -227. 8. Gargosky, S. E., Wallace, J. C., Upton, F. M. & Ballard, F. J. (1987) Biochem. J . 247, 427-432. 9. Carver, J . A. (1987) Eur. J . Bioclwm. 168, 193-199. 10. Carver, J. A. (1988) Biochem. Biophys. Res. Commun. 150, 552560. 11. Di Bello, C., Gozzini, L., Tonellato, M., Grdzia Corradini, M., D’Auria, G., Paolillo, L. & Trivellone, E. (1988) FEBS Letr. 237, 85 -90. 12. Erne, D. & Schwyzer, R. (1987) Biochemi,stry 26, 6316-6319. 13. Cavatorta, P.. Farruggia, G., Masotti, L.; Sartor, G . & Szabo. A. G. (1 986) Biochem. Biophys. Res. Commun. 141, 99 - 105. 14. Rance, M., Sarensen, 0. W., Bodenhausen, G., Wagner, G., Ernst, R. R. & Wiithrich, K . (1983) Biochem. Biophys. Res. Commun. 117,479-485. 15. States, D. J., Haberkorn, R. A. & Ruben, D. J. (1982) J . Mugn. Reson. 48, 286-292. 16. Wiithrich, K., Wider, G., Wagner, G. & Braun, W. (1982) J . Mol. Biill. 155, 311-319. 27. Wiithrich, K. (1986) N M R of proteins and nucleic ucids, Wiley, New York. 18. Wuthrich, K., Billeter, M. & Brdun, W. (1984) J . Mol. Biol. 180, 71 5 - 740. 19. Wagner, G., Neuhaus, D., Worgotter, E., Vasik, M., Kagi, J. H. R. & Wiithrich, K. (1986) 1.MoL B i d . 187, 131 - 135. 20. Tappin, M. J., Pastore, A,, Norton, R. S., Freer, J. H . &Campbell, I. D. (1988) Biochemistry 27, 1643-1647. 21. Pardi, A , , Billeter, M. & Wiithrich, K. (1984) J . Mol. Biol. 180, 141 - 751. 22. Zuiderweg, E. R. P., Boelens, R. & Kaptein, R. (1985) Biopolymws 24, 601 - 61 1. 23. Esposito, G., Carver, J. A., Boyd, J. & Campbell, 1. D. (1987) Bioclwnistry 26, 1043 1050. 24. Groncnborn, A. M., Bovermann, G. &Clore, G. M. (1987) FEBS Lett. 215, a8 -93. 25. Bazzo, R . , Tappin, M . .I.,Pastore, A,, Harvey, T. S., Carver, J . A. &Campbell, I. D. (1988) Eur. J . Biochem. 173, 139-146. 26. Shoemaker, K. R., Kim, P. S., York, E. J., Stewart, J. M. & Baldwin, R. L. (1987) Nuiure 326, 563 - 567. 27. Chou, P. Y. & Fasman, G. D. (1978) Adv. Enzymol. 47,45 - 148. 28. Rico, M., Santoro, J., Bermejo, F. J., Herranz, J., Nieto, J. L., Gallego, E. & Jimenez, M. A. (1986) Biopolymers 25, 1031 1053. 29. Richardson, J. S. & Richardson, D. C. (1988) Science 240,16481652. 30. Presta, L. G. & Rose, G. D. (1988) Science 240, 1632-1641. 31. Coy, D. H., Heinz-Erian, P., Jiang, N-Y., Sasaki, Y., Taylor, J., Moureau, J.-P., Wolfrey, W. T., Gardner, J. D. & Jensen, R.T. (1988) J. Biol. Chem. 263, 5056-5060. 32. Rivier, J. E. &Brown, M. R. (1978) Biochemistry 17,1166-1771. 33. Schwyzer, R. (1986) Biochemistry 25, 6335 -6342. 34. Briggs, M. S . . Cornell, D. G., Dluhy. R. A. & Gierasch, L. M. (1986) Science 233,206- 208. -