A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1.
Electrophysiological characterisation of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into defined lipid bilayers. Here, we describe an automated technique for reconstitution of membrane proteins into lipid bilayer membranes, which substantially reduces both the reconstitution time and the amount of protein required for the membrane formation. The method allows the investigation of single protein channels as well as insertion of multiple copies (approximately 10(7)) into a single bilayer. Despite a comparatively large membrane area (up to 300 microm diameter), the high stability of the membrane permits the application of transmembrane voltages up to 300 mV. This feature is especially important for studies of inner membrane mitochondrial proteins, since they act at potentials up to approximately 200 mV under physiological conditions. It is a combination of these advantages that enables the detailed investigation of the minuscule single protein conductances typical for proton transporters. We have applied the new technique for the reconstitution and electrophysiological characterisation of human recombinant uncoupling protein 1, hUCP1, that has been overexpressed in E. coli and purified from inclusion bodies. We demonstrate that hUCP1 activity in the presence of fatty acids is comparable to the activity of UCP1 isolated from brown adipose tissue.
Beck
Valeri
V
Institute of Cell Biology and Neurobiology, Charité Universitätsmedizin, Schumannstr. 20/21, 10098 Berlin, Germany.
Jabůrek
Martin
M
Breen
Eamon P
EP
Porter
Richard K
RK
Jezek
Petr
P
Pohl
Elena E
EE
D002352:Carrier Proteins / Q000737:chemistry*; D005227:Fatty Acids / Q000737:chemistry; D006096:Gramicidin / Q000737:chemistry; D006801:Humans; D007473:Ion Channels; D008051:Lipid Bilayers / Q000737:chemistry*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry*; D024101:Mitochondrial Proteins; D018408:Patch-Clamp Techniques; D017494:Proton Pumps / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D005227:Fatty Acids; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D017494:Proton Pumps; D011994:Recombinant Proteins; C000604371:UCP1 protein, human; D000071256:Uncoupling Protein 1; D006096:Gramicidin