Show simple item record

dc.contributor.authorMitra, Joydeep
dc.date.accessioned2018-07-12T18:50:46Z
dc.date.available2018-07-12T18:50:46Z
dc.date.issued1997
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 58-03, Section: B, page: 1160.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9724864
dc.identifier.urihttps://hdl.handle.net/20.500.12202/3716
dc.description.abstractSomatostatin (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.
dc.publisherProQuest Dissertations & Theses
dc.subjectBiophysics.
dc.subjectMolecular biology.
dc.subjectCellular biology.
dc.titleIdentification of structural domains in peptide hormone precursors
dc.typeDissertation


Files in this item

FilesSizeFormatView

There are no files associated with this item.

This item appears in the following Collection(s)

Show simple item record