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A Close-To-Native System to Study Membrane Protein Dynamics by Single-Molecule Optical Microscopy: an Application to Bacteriorhodopsin

Published online by Cambridge University Press:  02 July 2020

Nicoletta Kahya ,
Eve I. Pécheur ,
Douwe A. Wiersma  and
Dick Hoekstra
Nicoletta Kahya
Affiliation:
Ultrafast Laser and Spectroscopy Laboratory, Optical Sciences, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Eve I. Pécheur
Affiliation:
Department of Membrane Cell Biology, University of Groningen, Antonius Deusinglaan 1, 9713, AVGroningen, The Netherlands
Douwe A. Wiersma
Affiliation:
Ultrafast Laser and Spectroscopy Laboratory, Optical Sciences, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Dick Hoekstra
Affiliation:
Department of Membrane Cell Biology, University of Groningen, Antonius Deusinglaan 1, 9713, AVGroningen, The Netherlands
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Abstract

Biological membranes are not just a passive component of the cells, they actively support their functioning as the imbedded protein machineries carry out a wide range of crucial biochemical processes. Although studies in vivo are becoming more and more accessible to single-molecule optical microscopy, in vitro studies are still very much informative for the understanding of individual biological machineries. One of the major goals is to define the minimum number of components of a machinery that is necessary for a particular step, thereby allowing detailed studies of the mechanism of action. Reconstitution of a transmembrane protein system in artificial membranes (liposomes) is the main method for such a strategy, which thus may provide the option to investigate the functioning of transport proteins, ion channels, fusion machineries, and signal transducers in relation to their environment.

We present a novel procedure in order to reconstitute transmembrane proteins in chemically well-defined and close-to-native lipid bilayers, providing an in vitro system for single-molecule optical microscopy. Furthermore, an application of this technique is shown in the case of a single-molecule study of protein-protein and protein-lipid interactions for the light-induced proton pump bacteriorhodopsin.

In this study, Giant Unilamellar Vesicles (GUV), 10-100 μm sized, are used as lipid bilayer models for several reasons.

Type
Bridging the Gap Between Structural and Molecular Biology (Organized by B. Herman)
Information
Microscopy and Microanalysis , Volume 7 , Issue S2: Proceedings: Microscopy & Microanalysis 2001, Microscopy Society of America 59th Annual Meeting, Microbeam Analysis Society 35th Annual Meeting, Long Beach, California August 5-9, 2001 , August 2001 , pp. 30 - 31
Copyright
Copyright © Microscopy Society of America 2001

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References

references

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