Identification of structural domains in peptide hormone precursors

Abstract

Somatostatin (SRIF) is a 14 amino acid hormone synthesized as a larger precursor, preproSRIF of {dollar}\sim{dollar}121 residues. I proposed that its structure facilitates its 'sorting' during secretion. To test this hypothesis, I overproduced anglerfish proSRIF-II in E.coli, purified it to homogeneity and used fluorescence spectroscopy, circular dichroism, {dollar}\sp1{dollar}H NMR and mass spectrometry to analyze its structure.;Purified proSRIF-II was mostly monomeric, although up to 15% formed dimers by interchain {dollar}\sp{lcub}91/102{rcub}{dollar}Cys-S-S-Cys{dollar}\sp{lcub}91/102{rcub}{dollar} linkage. Pure native proSRIF-II (conformer 'N') had an open and flexible conformation--its fluorescence (three Trp{dollar}\sp{lcub}21,36,96{rcub}{dollar} one Tyr{dollar}\sp{lcub}95{rcub}{dollar}) changed between 4 and 25{dollar}\sp\circ{dollar}C. Circular dichroism confirmed this observation, although it detected {dollar}\sim{dollar}22% {dollar}\alpha{dollar}-helicity, which was susceptible to treatments with {dollar}\ge{dollar}3 M urea or temperatures above 60{dollar}\sp\circ{dollar}C. {dollar}\sp1{dollar}H NMR at pH 5.5 to 8.5 reaffirmed proSRIF-II to have an open conformation with many exchangeable protons.;ProSRIF-II shifted from an open to an ordered conformation when its 'N' conformer was heated slowly to 85{dollar}\sp\circ{dollar}C and re-cooled to 4{dollar}\sp\circ{dollar}C, manifesting a 16-fold increase in {dollar}\alpha{dollar}-helicity (conformer 'H'). ProSRIF-II's CD spectrum, upon heating and re-cooling twice, was consistent with {dollar}\beta{dollar}-strand/sheet structure with {dollar}\beta{dollar}-turns (conformer 'B'). Mass spectrometry, HPLC and gel exclusion chromatography confirmed these conformational changes did not result from protein aggregation or heat-induced oligomerization.;By comparing the susceptibility of 'N' and 'H' proSRIF-II to limited trypsin digestion, I found that distinct regions of the prohormone were protease resistant in the 'H' conformer. Mass spectrometry of proSRIF-II tryptic digests showed Ser{dollar}\sp{lcub}28{rcub}{dollar}-Arg{dollar}\sp{lcub}39{rcub}{dollar} and Ala{dollar}\sp{lcub}89{rcub}{dollar}-Lys{dollar}\sp{lcub}92{rcub}{dollar} were protected in 'H' but not in 'N' proSRIF-II, suggesting that these domains were buried in the 'H' conformer. Furthermore, both 'N' and 'H' proSRIF-II were efficiently and accurately processed by prohormone convertase 1/3 (PC1/3) in vitro to yield physiological cleavage products.;Together, these data identify three conformers (N, H and B) and a novel protein folding pathway for proSRIF-II. 'B' proSRIF-II with {dollar}\beta{dollar}-strand/sheet and {dollar}\beta{dollar}-turns may be processed in vivo to generate mature somatostatin.