Quantitation of protein orientation in flow-oriented unilamellar liposomes by linear dichroism

Jascindra Rajendra, Angeliki Damianoglou, Matthew Hicks, Paula Booth, P. Mark Rodger, Alison Rodger*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)

Abstract

The linear dichroism of the visible wavelength transitions of retinal have been used to analyse linear dichroism spectra to determine the orientation of aromatic and peptide structural motifs of Bacteriorhodopsin incorporated into unilamellar soy bean liposomes. The results are consistent with the available X-ray data. This proves that visible light absorbing chromophores can be used to analyse linear dichroism data to give the orientation of membrane proteins in membrane mimicking environments. The work has been extended by screening a wide range of hydrophobic molecules with high extinction coefficients in transitions above 300 nm to find molecules that could be used as independent probes of liposome orientation for experiments involving proteins incorporated into liposomes. Three probes were found to have potential for future work: bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC4), retinol and rhodamine B. All three can be used to determine the orientation of the porphyrin of cytochrome c, the aromatic residues of gramicidin and the helices of both proteins. The orientation parameter, S, for the liposomes varied from batch to batch of unilamellar liposomes prepared by extruding through a 100 nm membrane. The value and variation in S was 0.030 ± 0.010. Repeat experiments with the same batch of liposomes showed less variation. Film LD data were measured for DiBAC4 and rhodamine B to determine the polarisations of their long wavelength transitions.

Original languageEnglish
Pages (from-to)210-220
Number of pages11
JournalChemical Physics
Volume326
Issue number1
DOIs
Publication statusPublished - 11 Jul 2006
Externally publishedYes

Keywords

  • Bacteriorhodopsin
  • Linear dichroism
  • Liposomes
  • Orientation
  • Probe molecules
  • Proteins

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