Binding Mechanism of Purine Nucleotides to Mitochondrial Uncoupling Proteins Explored by Recognition Force Spectroscopy
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60th Annual Meeting of Biophysical Society
Regulated transport of protons across the inner mitochondrial membrane is essential for physiological processes such as ATP synthesis and heat production. Besides the involvement in thermogenesis, uncoupling protein family members (UCP1-UCP3) were proposed to regulate reactive oxygen species (ROS) and cell metabolism . Although it is accepted that UCP activity is inhibited by purine nucleotides, the exact molecular mechanism of inhibition and binding is still unclear. Previously, we hypothesized that PNs bind to UCP1 from cis- and trans-side, although only cis-binding led to protein inhibition . In this work we aimed to elucidate the binding of various nucleotides to UCP1-UCP3 on the single-molecule level. For this we reconstituted UCPs in bilayer lipid membranes at low density and probed the interaction forces between UCP and PNs coupled to AFM cantilevers using recognition force spectroscopy . Our studies revealed that the life time of the UCP-PN bond tended to increase with the phosphorylation degree of the nucleotide. However comparison between UCP1, UCP2 and UCP3 revealed different strengths of binding. Moreover, mutations of three arginine residues (R276L, R83T, R182Q) known to be crucial for nucleotide binding were studied to reveal a detailed picture about the binding mechanism of the nucleotides to the UCPs on a structural level.