Biphasic Proton Transport Mechanism for Uncoupling Proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Protein Function

Background:

• Uncoupling proteins (UCPs) mediate proton transport across membranes via conformational state changes.
• Salt-bridge networks in UCPs are critical for regulating these cytoplasmic and matrix states.
• Understanding UCP2's mechanism is key to elucidating its role in cellular energy regulation.

Purpose of the Study:

• To investigate the role of the matrix salt-bridge network in UCP2 proton transport.
• To elucidate the structural and functional mechanisms of proton transport in tetrameric UCP2.
• To propose a molecular model for UCP2 proton transport and its regulation.

Main Methods:

• Site-directed mutagenesis to create UCP2 mutants targeting matrix salt-bridges.
• Recombinant protein expression in E. coli membranes and reconstitution into lipid bilayers.
• Analysis using gel electrophoresis, circular dichroism, fluorescence assays, and molecular dynamics simulations.

Main Results:

• Identified five salt-bridges in the UCP2 matrix network, more than previously reported.
• Demonstrated that the matrix network regulates proton transport by stabilizing helix proximity and limiting activator binding.
• Proposed a biphasic two-state model for proton transport in functional tetrameric UCP2.
• Showed ATP inhibits UCP2 by occluding the pore via interactions with matrix network residues.

Conclusions:

• The UCP2 matrix network is crucial for regulating proton transport and conformational state transitions.
• A detailed molecular model for UCP2 proton transport and purine nucleotide inhibition is proposed.
• Findings provide novel insights into the complex mechanism of UCP-mediated proton transport.