title: Synthesis of an iberiotoxin derivative by chemical ligation: a method for improved yields of cysteine-rich scorpion toxin peptides.
abstract: Automated and manual solid phase peptide synthesis techniques were combined with chemical ligation to produce a 37-residue peptide toxin derivative of iberiotoxin which contained: (i) substitution of Val(16) to Ala, to facilitate kinetic feasibility of native chemical ligation, and; (ii) substitution of Asp(19) to orthogonally protected Cys-4-MeOBzl for chemical conjugate derivatization following peptide folding and oxidation. This peptide ligation approach increased synthetic yields approximately 12-fold compared to standard linear peptide synthesis. In a functional inhibition assay, the ligated scorpion toxin derivative, iberiotoxin V16A/D19-Cys-4-MeOBzl, exhibited 'native-like' affinity (K(d)=1.9 nM) and specificity towards the BK Ca(2+)-activated K(+) Channel (K(Ca)1.1). This was characterized by the rapid association and slow dissociation rates (k(on)=4.59 x 10(5)M(-1)s(-1); k(off)=8.65 x 10(-4) s(-1)) as determined by inhibition of macroscopic whole-cell currents of cloned human K(Ca)1.1 channel. These results illustrate the successful application of peptide chemical ligation to improve yield of cysteine-rich peptide toxins over traditional solid phase peptide synthesis. Native chemical ligation is a promising method for improving production of biologically active disulfide containing peptide toxins, which have diverse applications in studies of ion-channel function.
authors.affiliation: Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI 96822, USA. jbingham@hawaii.edu
authors.forename: Jon-Paul
authors.initials: JP
authors.lastname: Bingham
authors.forename: Joycelyn B
authors.initials: JB
authors.lastname: Chun
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Ruzicka
authors.forename: Qing X
authors.initials: QX
authors.lastname: Li
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Tan
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Kaulin
authors.forename: Darren R
authors.initials: DR
authors.lastname: Englebretsen
authors.forename: Edward G
authors.initials: EG
authors.lastname: Moczydlowski
mesh_terms: D002460:Cell Line; D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology; D017510:Protein Folding; D012604:Scorpion Venoms / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D051036:Large-Conductance Calcium-Activated Potassium Channels; D010446:Peptide Fragments; D010455:Peptides; D012604:Scorpion Venoms; C064719:iberiotoxin; D003545:Cysteine