title: cDNA sequence and in vitro folding of GsMTx4, a specific peptide inhibitor of mechanosensitive channels.
abstract: The peptide GsMTx4 from the tarantula venom (Grammostola spatulata) inhibits mechanosensitive ion channels. In this work, we report the cDNA sequence encoding GsMTx4. The gene is translated as a precursor protein of 80 amino acids. The first 21 amino acids are a predicted signal sequence and the C-terminal residues are a signal for amidation. An arginine residue adjacent to the N-terminal glycine of GsMTx4 is the cleavage site for release. The resulting peptide is 34 amino acids in length with a C-terminal phenylalanine and not a serine-alanine previously identified [J. Gen. Physiol. 115 (2000) 583]. We chemically synthesized this peptide and folded it in 0.1 M Tris, pH 7.9 with oxidized/reduced glutathione (1/10). Properties of the synthetic peptide were identical to the wild type for high performance liquid chromatography (HPLC), mass spectrometry, CD, and NMR. We also cloned GsMTx4 in a thioredoxin fusion protein system containing six histidines. Nickel affinity columns allowed rapid purification and folding occurred in conditions described above with 0.5 M guanidiniumHCl present. Thrombin cleavage liberated GsMTx4 with three extra amino acids at the N-terminus. The retention time in HPLC analysis and the CD spectrum was similar to wild type. Both the synthetic and cloned peptides were active in the patch clamp assay.
authors.affiliation: Center for Single Molecule Biophysics, SUNY at Buffalo, 320 Cary Hall, Buffalo, NY 14214, USA.
authors.forename: Kimberly Laskie
authors.initials: KL
authors.lastname: Ostrow
authors.forename: Aaron
authors.initials: A
authors.lastname: Mammoser
authors.forename: Tom
authors.initials: T
authors.lastname: Suchyna
authors.forename: Frederick
authors.initials: F
authors.lastname: Sachs
authors.forename: Robert
authors.initials: R
authors.lastname: Oswald
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kubo
authors.forename: Naoyoshi
authors.initials: N
authors.lastname: Chino
authors.forename: Philip A
authors.initials: PA
authors.lastname: Gottlieb
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000037:antagonists & inhibitors*; D040542:Mechanotransduction, Cellular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding; D012333:RNA, Messenger / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013111:Spider Venoms / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D036341:Intercellular Signaling Peptides and Proteins; D007473:Ion Channels; C411249:MTx4 protein, Grammostola spatulata; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013111:Spider Venoms