Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment

Archives of Microbiology
Busaba Powthongchin, Chanan Angsuthanasombat

Abstract

The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis was employed to investigate the contributions to haemolysis of five predicted helices within the N-terminal hydrophobic region of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1(500-522), 2(529-550), 3(571-593) and 5(678-698), respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in the predicted helix 4(602-627) (A616P). MALDI-TOF-MS and LC-MS-MS analyses verified the palmitoylation at Lys983 of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation was not a...Continue Reading

References

May 21, 1992·Nature·S ChoeD Eisenberg
Jan 14, 1991·FEBS Letters·I E EhrmannE L Hewlett
Mar 1, 1991·Molecular Microbiology·R A Welch
Jan 5, 1989·Nature·M W ParkerD Tsernoglou
May 5, 1982·Journal of Molecular Biology·J Kyte, R F Doolittle
Dec 1, 1994·Computer Applications in the Biosciences : CABIOS·M G Claros, G von Heijne
Aug 1, 1996·Protein Science : a Publication of the Protein Society·B RostR Casadio
Mar 15, 2000·Journal of Molecular Biology·W P Russ, D M Engelman
May 24, 2000·Proceedings of the National Academy of Sciences of the United States of America·M EilersP J Fleming
Oct 9, 2001·Bioinformatics·G E Tusnády, I Simon
Aug 31, 2002·Biophysical Journal·Patrick C A van der WelRoger E Koeppe
Apr 5, 2003·The Journal of Biological Chemistry·Petra MalovrhGregor Anderluh
Oct 8, 2004·Journal of Biochemistry and Molecular Biology·Chanan AngsuthanasombatSakol Panyim
Nov 25, 2004·Progress in Biophysics and Molecular Biology·Michael W Parker, Susanne C Feil
Mar 28, 2006·Current Opinion in Structural Biology·Sarah J Tilley, Helen R Saibil
Mar 10, 2007·The Journal of Biological Chemistry·Marek BaslerRadim Osicka

❮ Previous
Next ❯

Citations

May 6, 2015·Toxins·Chattip KurehongChanan Angsuthanasombat
Sep 2, 2015·Biochemical and Biophysical Research Communications·Riyaz Ahmad PanditChanan Angsuthanasombat
Mar 23, 2011·Toxicon : Official Journal of the International Society on Toxinology·Chattip KurehongChanan Angsuthanasombat
Sep 13, 2015·Toxicon : Official Journal of the International Society on Toxinology·Sirikran JuntapremjitChanan Angsuthanasombat

❮ Previous
Next ❯

Related Concepts