The conformational properties of somatostatin. IV. The conformers contributing to the conformation equilibrium of somatostatin in aqueous solution as found by semi-empirical energy calculations and high-resolution NMR experiments.

The results of a conformational study by 1H and 13C high-resolution NMR at 270 and 500 MHz on the peptide hormone somatostatin have been compared with a series of conformers generated by semi-empirical energy calculations. The use of specifically deuterated phenylalanine residues has enabled us to confirm and supplement the identification of all but the phenylalanine aromatic resonances in the proton spectra of somatostatin. In order to minimize the risk of overlooking some low-energy conformations, four different strategies have been used for the generation of the conformers: two based on combinations of conformations of fragments that had been studied before, one on a random procedure and one on the conformational constraints existing in bicyclic analogs with high biological activity. The experimental values of 3JNH-C alpha H and 3J alpha beta coupling constants and the existence of several ring current shifts allowed us to select from the calculations those families of low-energy conformers that are compatible with the NMR results. The NH temperature coefficients do not warrant the existence of any stable beta or gamma turns in the molecule, although the region SRIF8-12 seems to be the most stable in this respect. In addition there are several upfield shifts: 0.2-0.4 ppm on the Lys9 side-chain, 0.3-0.5 ppm on the Phe6 alpha, beta and Phe7 alpha protons, as well as some 0.2-0.3 ppm shifts on parts of two phenylalanine ring systems. Almost all of these shifts decrease considerably with increasing temperature. Most of the observed NMR results are compatible with the properties of one family of low-energy conformations whose main features are a double beta II bend Trp8-Lys9, Thr10 -Phe11, a close proximity of the Trp8 and Lys9 side chains and an orientation of Phe7 towards the Phe6 alpha, beta protons. We conclude that this set of conformations forms a major contribution to the conformational equilibrium at room temperature. The properties of this and several other sets of low-energy conformations that do not dominate in aqueous solution are discussed in relation to al available experimental evidence.