The Orientation of Nisin in Membranes
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- 作者：Eefjan Breukink ; Cindy van Kraaij ; Annemieke van Dalen ; Rudy A. Demel ; Roland J. Siezen ; Ben de Kruijff ; and Oscar P. Kuipers
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：June 2, 1998
- 年：1998
- 卷：37
- 期：22
- 页码：8153 - 8162
- 全文大小：120K
- 年卷期：v.37,no.22(June 2, 1998)
- ISSN：1520-4995
文摘
Nisin is a 34 residue long peptide belonging to the group Alantibiotics with antimicrobialactivity against Gram-positive bacteria. The antimicrobialactivity is based on pore formation in thecytoplasmic membrane of target organisms. The mechanism whichleads to pore formation remains tobe clarified. We studied the orientation of nisin viasite-directed tryptophan fluorescence spectroscopy.Therefore, we engineered three nisin Z variants with uniquetryptophan residues at positions 1, 17, and32, respectively. The activity of the tryptophan mutants againstGram-positive bacteria and in modelmembrane systems composed of DOPC or DOPG was established to be similarto that of wild type nisinZ. The tryptophan fluorescence emission maximum showed anincreasing blue-shift upon interactionwith vesicles containing increased amounts of DOPG, with the largesteffect for the 1W peptide. Studieswith the aqueous quencher acrylamide showed that all tryptophans becameinaccessible from the aqueousphase in the presence of negatively charged lipids in the vesicles.From these results it is concluded thatanionic lipids mediate insertion of the tryptophan residues in at leastthree positions of the molecule intothe lipid bilayer. The depth of insertion of the tryptophanresidues was determined via quenching of thetryptophan fluorescence by spin-labeled lipids. The results showedthat the depth of insertion was dependenton the amount of negatively charged lipids. In membranescontaining 50% DOPG, the distances fromthe bilayer center were determined to be 15.7, 15.0, and 18.4 Å forthe tryptophan at position 1, 17, and32, respectively. In membranes containing 90% DOPG, thesedistances were calculated to be 10.8, 11.5,and 13.1 Å, respectively. These results suggest an overallparallel average orientation of nisin in themembrane, with respect to the membrane surface, with the N-terminusmore deeply inserted than theC-terminus. These data were used to model the orientation of nisinin the membrane.