Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold.
Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg(50) residue to generate a smaller 49-residue mature peptide. The Arg(50) cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly alpha-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.
Cummins
Scott F
SF
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-0620, USA. sfcummin@utmb.edu
Xie
Fang
F
Misra
Milind
M
Amare
Andinet
A
Jakubowski
Jennifer A
JA
de Vries
Melissa R
MR
Sweedler
Jonathan V
JV
Nagle
Gregg T
GT
Schein
Catherine H
CH
D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010675:Pheromones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D004220:Disulfides; D010675:Pheromones; D011994:Recombinant Proteins