Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes
Artikel i vetenskaplig tidskrift, 2007

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.

reconstitution

membrane

immobilization

qcm-d

nicotinamide nucleotide transhydrogenase

vesicles

lipid-bilayers

surface-plasmon resonance

escherichia-coli

dna

Författare

Annette Graneli

Göteborgs universitet

Jason J. Benkoski

Analytical Biochemistry

0003-2697 (ISSN) 1096-0309 (eISSN)

Vol. 367 87-94

Ämneskategorier

Biokemi och molekylärbiologi

Analytisk kemi

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

DOI

10.1016/j.ab.2007.03.031