somatostatin-28: a conformational analysis
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Soma tosta tin-28: A Conforma tional Analysis
The cyclic tetradecapeptide somatostatin can be found in hypothalamic and pancreatic extracts of various Somatostatin-like immunoreactivity has also been observed in materials of higher molecular weight, suggesting the presence of pro-hormonal forms of this molecule.- The isolation and primary structure of an N-terminally extended form of porcine somatostatin has been reported re~ent ly .~ It contains 28 amino acid residues and has been called somatostatin-28. The primary structure of this new peptide hormone is the following:
H-Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys- 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4
Ala-Gly-Cls-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-C~s-OH 15 16 17 18 19 20 21 22 23 24 25 26 27 28
This structure was proven by total synthesis and comparative analysis of the synthetic and the natural product by means of chromatographic, immunological, and biological assays.10
Symbols for amino acids are according to IUPAC-IUB recommendations." Other ab- breviations are as follows: Soma-28, somatostatin-2d; Soma-14, somatostatin-14; and [l-141, N-terminal tetradecapeptide of somatostatin-28.
This paper describes the results of a conformational study on synthetic Soma-28 using CD measurements as the first approach to its structural properties in aqueous solution.
Soma-28 and Soma-14 were synthesized as described previously.10*12 The synthesis and characterization of fragment [ 1-14] will be reported elsewhere.
Fig. 1. CD spectra of Soma-28 (curve l), Soma-14 (curve 2), and [l-141 (curve 3) in aqueous solution in the 310-220-nm absorption region and (a) in the 350-250-nm absorption region. The peptide concentrations were 2.7 X 10-5M, 4.9 X 10-5M, and 4.9 X 10-5M, respec- tively.
Biopolymers, Vol. 20, 1741-1745 (1981) 0 1981 John Wiley & Sons, Inc. CCC 0006-3525/81/081741-05$01.00
1742 BIOPOLYMERS VOL. 20 (1981)
I I I I I I
250 240 230 220 210 200 190 h ( n m )
Fig. 2. CD spectra of Soma-28 (curve l ) , Soma-14 (curve 2), and [l-141 (curve 3) in aqueous solution in the 250-190-nm absorption region. The concentrations of the peptides were the same as in Fig. 1.
CD measurements were performed a t 25°C using a Mark I11 Jobin-Yvon Dichrograph (Instruments S.A. Paris), equipped with a Nicolet B-NC 12 signal-averaging system. The number of accumulated scans ranged from 100 to 150 depending on the intensity of the CD signal. Averaging of the accumulated spectra was performed using the Nicolet Lab-80 General Signal Averaging Package. All spectra reported in this paper are original, computer-drawn CD curves. In the figures, [O]M represents ellipticity values (deg cm2 mol-') per mole of peptide.
The near-uv CD spectra of Soma-28, Soma-14, and [l-141 in aqueous solutions are reported in Fig. l(a). The octacosapeptide (curve 1) is characterized by negative bands located at 292, 285,267, and 265 nm, with molar ellipticities all around 1000 deg cm2 mol-'. These hands arise from the contributions of the Lb transitions of the Phe residues at positions 20,21,25, of the La and Lb transition of Trp at position 22, and of the disulfide group. The low ellipticity values and the shape of the spectrum do not indicate the presence of specific steric constraints for the Phe and Trp side chains, which should exhibit a high degree of conformational freedom. All chromophores absorbing in the near-uv are located in the C-terminal region of Soma-28, i.e., in the Soma-14 moiety. These represent a unique tool for comparing the conformational states of the corresponding sequence in the two peptides. Soma-28 and Soma-14 exhibit almost identical near-uv CD properties [Fig. l(a)]. We therefore assume that no appreciable conformational modification of the Soma-14 moiety takes place on elongating its N-terminal sequence to obtain Soma-28. Since a stable ring conformation has been suggested for the former in aqueous ~ o l u t i o n , ' ~ ~ ' ~ the same conformation should be predominant in the C-ter- minal region of the latter.
The far-uv CD spectra of Soma-28, Soma-14, and [l-141 are reported in Figs. 1 and 2. The
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250 240 230 220 210 200 190 A l n m l
Fig. 3. CD spectrum of Soma-28 (curve 1) and sum of the normalized spectra of Soma-14 and [l-141 (curve 2) in the 250-190-nm absorption region (top) and differential spectrum (curve 2 - curve 1) (bottom).
spectrum of Soma-28 (curve 1) is characterized by a negative shoulder around 230 nm and by a negative minimum located at 198 nm, with molar ellipticity of -4.2 X 105 deg cm2 mol-' (-15,000 units/residue). These figures are consistent with the presence of a predominantly aperiodic structure in the linear region of the hormone and match well with those previously reported for gastrointestinal peptides of similar molecular size.I5 Consistent with previously reported result^,'^ the C-terminal cyclic peptide exhibits peculiar CD properties (curves 2 in Figs. 1 and 2), with a positive absorption at 226 nm and a weak negative band around 200 nm (molar ellipticity, -1.1 X lo5 units). On the other hand, the overall spectral features of the linear [l-141 sequence are similar to those of Soma-28 in the peptide absorption region. Indeed, the tetradecapeptide exhibits a negative shoulder around 225-230 nm and a negative minimum at 197 nm, with molar ellipticity of -2.8 X lo5 units (-20,000 unitshesidue) (curve 3 in Figs. 1 and 2).
In order to evaluate the conformational effects due to the presence of the cyclic tetrade- capeptide bound to [I-141 in Soma-28, it is interesting to compare the CD pattern of the latter with a simulated spectrum calculated by the addition of the normalized spectra of Soma-14 and [l-141 in the same solvent. The results are presented in Fig. 3. Although curves 1 (Soma-28) and 2 (Soma-14 + [l-14)) closely resemble each other, small but significant changes can be observed. The differential spectrum (also reported in Fig. 3) shows two minima at 225 and 198 nm, the intensities of which are well above the experimental error.
The following possibilities may explain the observed CD differences: (1) The curve com- puted from the spectra of the separate fragments could be affected by nonnegligible contri-
1744 BIOPOLYMERS VOL. 20 (1981)
butions of the chain ends which are no longer present in the combined peptide (i.e., the C- terminus of [l-141 and the N-terminus of Soma-14). The curve should also be somewhat underestimated around 200 nm, due to neglecting the contribution of the new peptide bond which is formed. (2) The conformation of the fragment(s) could change as a consequence of bond formation between the above-mentioned end groups.
Since in a denaturating solvent such as 6M guanidine hydrochloride the spectrum of the octacosapeptide and the sum of its components match very well from 350 to 215 nm (tran- sparence limit of the samples), the first possibility seems to be ruled out at least for the 225-nm difference band. The conformational state of the separate fragments appears, therefore, slightly changed in Soma-28.
As the near-uv measurements [Fig. l(a)] strongly suggest that the ring conformation of Soma-14 is retained in the C-terminal sequence of Soma-28, the structural modification should be essentially located in the linear region of the hormone.
The above results are possibly related to the presence of intramolecular interactions between the linear and the cyclic region of Soma-28, intermolecular effects being rather unlikely, due to the very low peptide concentration used in this work (c Q 3 X 10-5M). On the other hand, the relatively small change between the computed and the observed spectra of Soma-28 in- dicates that the equilibrium of the conformers is only slightly modified when the [l-141 peptide is bound to Soma-14. Thus, the above-mentioned interactions appear to be rather weak in aqueous solution. Accordingly, the conformation of the cyclic moiety of Soma-28 is not ap- preciably affected by the presence of the (1-141 sequence.
Preliminary measurements in trifluoroethanol also point towards the onset of an interaction between the N-terminal and the C-terminal sequences in the Soma-28 molecule. Nmr studies are required to identify the amino acid residues involved in the process.
A final consideration is related to the biologically relevant question of whether or not Soma-28 is only a pro-horponal form of Soma-14. Our findings suggest that the [l-141 se- quence may also play a role in the hormone function, so that Soma-28 might itself exhibit specific biological activity.
References
1. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J. & Guillemin, R. (1973)
2. Brazeau, P., Vale, W., Burgus, R. & Guillemin, R. (1974) Can. J. Biochem. 52,1067-
3. Schally, A., Dupont, A., Arimura, A., Redding, T., Nishi, N., Linthicum, G. &
4. Spiess, J., Rivier, J., Rodkey, J., Bennet, C. & Vale, W. (1979) Proc. Natl. Acad. Sci.
5. Noe, B., Spiess, J., Rivier, J. & Vale, W. (1979) Endocrinology 105,1410-1415. 6. Arimura, A., Sato, H., Dupont, A., Nishi, N. & Schally, A. (1975) Science 189,1007-
7. Millar, R. P. (1978) J. Endocrinol. 77,429-430. 8. Millar, R. P., Denniss, P., Tobler, C., King, J. C., Schally, A. & Arimura, A. (1979) in
Biologie Cellulaire des Processus Neuroshetoires Hypothalamiques, Vincent, J. D. & Kordon, C., Eds., CNRS, Paris.
Science 179,77-79.
1072.
Schlesinger, D. (1976) Biochemistry 15,509-514.
USA 76,2974-2978.
1009.
9. Pradayrol, L., Jornvall, H., Mutt, V. & Ribet, A. (1980) FEES Lett. 109,55-58. 10. Wunsch, E., Moroder, L., Gemeiner, M., Jaeger, E., Ribet, A., Pradayrol, L. & Vaysse,
11. IUPAC-IUB Commission (1966) Arch. Biochem. Biophys. 115,l-12. 12. Moroder, L., Gemeiner, M., Goehring, W., Jaeger, E., Thamm, P. & Wunsch, E. (1981)
N. (1980) 2. Naturforsch., Teil B 35,911-919.
Biopolymers 20,17-37.
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13. Holladay, L. A. & Puett, D. (1976) R o c . Natl. Acad. Sci. USA 73,1199-1202. 14. Arison, B. H., Hirschmann, R. & Veber, D. F. (1978) Bioorg. Chen. 7,447-451. 15. Peggion, E., Jaeger, E., Knof, S., Moroder, L. & Wunsch, E. (1981) Biopolymers 20,
ERICH WUNSCH ERNST JAEGER LUIS MORODER
633-652.
Max-Planck Institut fur Biochemie Abteilung fur Peptidchemie D-8033 Martinsried, Federal Republic of Germany
EVARISTO PEGGION MANLIO PALUMBO
Centro di Studi sui Biopolimeri Istituto di Chimica Organica 1-35100 Padova, Italy
Received December 8,1980 Accepted March 25,1981