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Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes

Graneli, Annette; Benkoski, Jason J and Höök, Fredrik LU (2007) In Analytical Biochemistry 367(1). p.87-94
Abstract
Surface analytical tools have gained interest in the bioanalytical field during recent years because they offer the possibility of more detailed investigations of biomolecular interactions. To be able to use such tools, the biomolecules of interest must be immobilized to a surface in a functioning way. For small water-soluble biomolecules, the surface immobilization is quite straightforward, but it has been shown to be difficult for large transmembrane proteins. In those cases, the solid surface often has a negative influence on the function of the transmembrane proteins. In this article, we present a new approach for surface immobilization of transmembrane proteins where the proteins were immobilized on a surface in a proteoliposome... (More)
Surface analytical tools have gained interest in the bioanalytical field during recent years because they offer the possibility of more detailed investigations of biomolecular interactions. To be able to use such tools, the biomolecules of interest must be immobilized to a surface in a functioning way. For small water-soluble biomolecules, the surface immobilization is quite straightforward, but it has been shown to be difficult for large transmembrane proteins. In those cases, the solid surface often has a negative influence on the function of the transmembrane proteins. In this article, we present a new approach for surface immobilization of transmembrane proteins where the proteins were immobilized on a surface in a proteoliposome multilayer structure. The surface-binding events and the structure of the surface-immobilized proteoliposomes were monitored using both the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. With this multilayer proteoliposome structure, it was possible to detect trypsin digestion of the transmembrane protein proton translocating nicotinamide nucleotide transhydrogenase in real time using SPR. The results from the combined SPR and QCM-D analysis were confirmed by fluorescence microscopy imaging of the multilayer structure and activity measurements of transhydrogenase. These results showed that the activity of transhydrogenase was significantly decreased in the bottom layer, but in the subsequent proteoliposome layers 90% of the activity was retained compared with bulk measurements. These results emphasize the importance of an immobilization strategy where the transmembrane proteins are lifted off the solid surface at the same time as the amount of protein is increased. We consider this new method for surface immobilization of transmembrane proteins to meet these demands and that the method will improve the possibility to use a variety of surface analytical tools for the analysis of interactions involving transmembrane proteins in the future. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
fluorescence microscopy, multilayers, cholesterol, DNA, proteoliposomes, biosensors, biomimetic synthesis
in
Analytical Biochemistry
volume
367
issue
1
pages
87 - 94
publisher
Elsevier
external identifiers
  • wos:000247868800010
  • scopus:34250619219
ISSN
1096-0309
DOI
10.1016/j.ab.2007.03.031
language
English
LU publication?
yes
id
f7de76f6-ac61-4939-ac8d-2829506d0c6f (old id 646009)
date added to LUP
2007-12-07 12:52:28
date last changed
2017-01-01 04:20:18
@article{f7de76f6-ac61-4939-ac8d-2829506d0c6f,
  abstract     = {Surface analytical tools have gained interest in the bioanalytical field during recent years because they offer the possibility of more detailed investigations of biomolecular interactions. To be able to use such tools, the biomolecules of interest must be immobilized to a surface in a functioning way. For small water-soluble biomolecules, the surface immobilization is quite straightforward, but it has been shown to be difficult for large transmembrane proteins. In those cases, the solid surface often has a negative influence on the function of the transmembrane proteins. In this article, we present a new approach for surface immobilization of transmembrane proteins where the proteins were immobilized on a surface in a proteoliposome multilayer structure. The surface-binding events and the structure of the surface-immobilized proteoliposomes were monitored using both the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. With this multilayer proteoliposome structure, it was possible to detect trypsin digestion of the transmembrane protein proton translocating nicotinamide nucleotide transhydrogenase in real time using SPR. The results from the combined SPR and QCM-D analysis were confirmed by fluorescence microscopy imaging of the multilayer structure and activity measurements of transhydrogenase. These results showed that the activity of transhydrogenase was significantly decreased in the bottom layer, but in the subsequent proteoliposome layers 90% of the activity was retained compared with bulk measurements. These results emphasize the importance of an immobilization strategy where the transmembrane proteins are lifted off the solid surface at the same time as the amount of protein is increased. We consider this new method for surface immobilization of transmembrane proteins to meet these demands and that the method will improve the possibility to use a variety of surface analytical tools for the analysis of interactions involving transmembrane proteins in the future.},
  author       = {Graneli, Annette and Benkoski, Jason J and Höök, Fredrik},
  issn         = {1096-0309},
  keyword      = {fluorescence microscopy,multilayers,cholesterol,DNA,proteoliposomes,biosensors,biomimetic synthesis},
  language     = {eng},
  number       = {1},
  pages        = {87--94},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes},
  url          = {http://dx.doi.org/10.1016/j.ab.2007.03.031},
  volume       = {367},
  year         = {2007},
}