The development of new methods and strategies for the investigation of membrane proteins is limited by poor solubility of these proteins in an aqueous environment and hindered by a number of other problems linked to the instability of the proteins outside lipid bilayers. Therefore, current research focuses on an analysis of membrane proteins incorporated into model lipid membrane, most frequently liposomes. In this work, we introduce a new electrochemical methodology for the analysis of transmembrane proteins reconstituted into a liposomal system. The proposed analytical approach is based on proteoliposomal sample adsorption on the surface of working electrodes followed by analysis of the anodic and cathodic signals of the reconstituted proteins. It works based on the fact that proteins are electroactive species, in contrast to the lipid components of the membranes under the given experimental conditions. Electroanalytical experiments were performed with two transmembrane proteins; the Na(+)/K(+)ATPase that contains transmembrane as well as large extramembraneous segments and the mitochondrial uncoupling protein 1, which is a transmembrane protein essentially lacking extramembraneous segments. Electrochemical analyses of proteoliposomes were compared with analyses of both proteins solubilized with detergents (C12E8 and octyl-PoE) and supported by the following complementary methods: microscopy techniques, protein activity testing, molecular model visualizations, and immunochemical identification of both proteins. The label-free electrochemical platform presented here enables studies of reconstituted transmembrane proteins at the nanomolar level. Our results may contribute to the development of new electrochemical sensors and microarray systems applicable within the field of poorly water-soluble proteins.

Department of Biophysics Centre of the Region Hana for Biotechnological and Agricultural Research Faculty of Science Palacky University Slechtitelu 27 783 71 Olomouc Czech Republic

Department of Medical Chemistry and Biochemistry Faculty of Medicine and Dentistry Palacky University Hnevotinska 3 775 15 Olomouc Czech Republic

Department of Pharmacology and Immunotherapy Veterinary Research Institute v v i Hudcova 70 621 00 Brno Czech Republic

Institute of Physics Academy of Sciences of the Czech Republic Na Slovance 2 18221 Prague Czech Republic

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Electrochemistry in sensing of molecular interactions of proteins and their behavior in an electric field

Lipid-based liquid crystalline materials in electrochemical sensing and nanocarrier technology