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Effect of Linker Length on Avidin Binding to Biotinylated Gramicidin A

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Abstract

Biotinylated gramicidins are an important component of the AMBRI® “ion channel switch™” biosensor. These gramicidin A (gA) analogues have a biotin attached to the C-terminus of gA via a number of aminocaproyl linker groups (X). The structure of gA5XB has been determined in deuterated sodium dodecyl sulfate micelles and is similar to native gA and other modified gA analogues. The biotin and aminocaproyl groups were mobile and located in the aqueous phase and when avidin was added, NMR and MS studies showed that gA5XB bound more effectively to avidin than gA2XB. The length and flexibility of the linker appears to be important for biotin–avidin binding and, in the AMBRI® biosensor, gA5XB is a more effective gated ion channel than gA2XB. The conformation and dynamics of the aminocaproyl linker groups were investigated using 2H solid-state NMR. Deuterated aminocaproyl linkers were coupled to gA and incorporated into oriented bilayers in order to analyse the order and dynamics of the aminocaproyl linker. The small 2H splittings and the T 1 relaxation times indicated that the aminocaproyl linker is undergoing fast rotation in phospholipid bilayers. Native d 4 -gA as well as d 4 -gA2XB, where the ethanolamine has been deuterated, were also incorporated into oriented bilayers. Solid-state 2H NMR data showed that the addition of the linker group restricted the mobility of the ethanolamine. However, these modifications to the C-terminus of gA did not interfere with ion channel function and clarify how the biotinylated gA analogues perform in the lipid bilayer as part of the AMBRI® biosensor.

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Abbreviations

Boc:

(tert-butyloxycarbonyl)

d 4 -gA:

gramicidin A with deuterated ethanolamine

fwhh:

full width half height

gA:

gramicidin A

gAnXB:

biotinylated gA with n aminocaproic acid linker groups

DHB:

2,5-dihydroxybenzoic acid

DMPC:

Dimyristoylphosphatidylcholine

MALDI MS:

Matrix-Assisted Laser Desorption/Ionization mass spectrometry

NMR:

nuclear magnetic resonance spectroscopy

RMSD:

root mean square deviation

SDS-d 25 :

perdeuterated sodium dodecyl sulfate

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Acknowledgments

AA wishes to thank Pacific Dunlop for award of a PhD scholarship and Dr Patrick C. A. van der Wel and Dr Denise Greathouse for help with the 2H NMR work.

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Authors and Affiliations

  1. School of Chemistry, University of Melbourne, 3010, Melbourne, VIC, Australia

    Aphrodite Anastasiadis, Craig J. Morton & Frances Separovic

  2. Biota Structural Biology Laboratory, St Vincents Institute, 3065, Melbourne, VIC, Australia

    Craig J. Morton

  3. School of Dental Science, University of Melbourne, 3010, Melbourne, VIC, Australia

    Gert H. Talbo

  4. Department of Chemistry & Biochemistry, University of Arkansas, Fayetteville, AR, 72701, USA

    Roger E. Koeppe II

  5. School of Chemistry, University of Melbourne, 3010, VIC, Australia

    Frances Separovic

Authors
  1. Aphrodite Anastasiadis
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  2. Craig J. Morton
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  3. Gert H. Talbo
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  4. Roger E. Koeppe II
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  5. Frances Separovic
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Correspondence to Frances Separovic.

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Australian Peptide Conference Issue.

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Anastasiadis, A., Morton, C.J., Talbo, G.H. et al. Effect of Linker Length on Avidin Binding to Biotinylated Gramicidin A. Int J Pept Res Ther 12, 243–252 (2006). https://doi.org/10.1007/s10989-006-9017-4

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  • DOI: https://doi.org/10.1007/s10989-006-9017-4

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