Study of Nucleotide Binding to the Uncoupling Protein 1 using Atomic Force Microscopy

Abstract

A tight proton transport regulation in the inner mitochondrial membrane is crucial for physiological processes such as ATP synthesis or non-shivering thermogenesis in the presence of uncoupling protein-1 (UCP1). Several hypotheses explaining the inhibitory effect of ATP on UCP were proposed, however, structural details are missing so far. Here we use the topographic and recognition (TREC) mode of an atomic force microscope to visualize UCP1 re-constituted into lipid bilayers (Beck, 2006) and to analyze the ATP-protein interaction on the single molecule level (Wildling, 2011). The comparison of recognition patterns obtained with anti-UCP antibody and ATP led to the conclusion that the ATP binding site can be accessed from both membrane sides. Using cantilever tips with different crosslinker lengths, we determined the location of the nucleotide binding site inside the membrane with 1 A precision. In the absence of the crystallographic structure for UCP1, these data provide valuable insight into the nucleotide binding mechanism.ReferencesBeck, V., Jaburek, M., Jezek, P., Pohl, E.E. (2006). A new automated technique for the reconstitution of hydrophobic proteins in planar bilayer membranes. Reconstitution of recombinant uncoupling protein 1. Biochim. Biophys. Acta (Bioenergetics), 1757(5-6):474-9.Wildling, L., Unterauer, B., Zhu, R., Rupprecht, A., Haselgruebler, T., Rankl, C., Ebner, A., Vater, D., Pollheimer, P., Pohl, E.E., Hinterdorfer, P., Gruber, H. J. (2011) Linking of sensor molecules with amino groups to amino-functionalized AFM tips. Bioconjugate Chem. 22, 1239-1248.