cDNA sequence and in vitro folding of GsMTx4, a specific peptide inhibitor of mechanosensitive channels.
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.
Ostrow
Kimberly Laskie
KL
Center for Single Molecule Biophysics, SUNY at Buffalo, 320 Cary Hall, Buffalo, NY 14214, USA.
Mammoser
Aaron
A
Suchyna
Tom
T
Sachs
Frederick
F
Oswald
Robert
R
Kubo
Shigeru
S
Chino
Naoyoshi
N
Gottlieb
Philip A
PA
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
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