DETERMINATION OF THE SOLUTION STRUCTURE OF THE PEPTIDE-HORMONE GUANYLIN - OBSERVATION OF A NOVEL FORM OF TOPOLOGICAL STEREOISOMERISM

Guanylin is a 15 amino acid mammalian hormone containing two disulfide bonds. Guanylin shares sequence similarity with the bacterial heat-st able enterotoxin (STa) and is capable of binding to and stimulating th e STa guanylyl cyclase receptor. Biologically active peptides have bee n prepared by two methods: (1) enzymatic treatment of a 99 residue pro protein (denoted proguanylin) expressed in Escherichia coli and (2) so lid-phase chemical synthesis. Although both sources yield material tha t is pure by high-performance liquid chromatography and mass spectrome try, analysis by nuclear magnetic resonance (NMR) indicates that pepti des from both sources contain two conformationally distinct species pr esent in a 1:1 ratio. The chemical shift differences between the two s pecies are large, allowing unambiguous sequential NMR assignments to b e made for both sets of resonances. Exchange between the two forms was not observed even at 70 degrees C. Structural restraints have been ge nerated from nuclear Overhauser effects and scalar coupling constants and used to calculate structures for both forms using distance geometr y and restrained energy minimization. The resulting structures for the first isoform are well defined (root-mean-square deviation from the a verage structure for backbone atoms of 0.47 Angstrom) and adopt a righ t-handed spiral conformation, similar to that observed for heat stable enterotoxin. The second isoform is less well defined (root-mean-squar e deviation from the average structure for backbone atoms of 1.07 Angs trom) but clearly adopts a very different fold consisting of a left-ha nd spiral. The differences in structure suggest that the two forms may have very different affinities toward the STa receptor. The observati on of such isomerism has important implications for the common practic e of introducing multiple disulfide bonds into small peptides to limit conformational flexibility and enhance bioactivity.