Membrane uncoupling protein 3 (UCP3), a member of the mitochondrial uncoupling protein family, was discovered in 1997. UCP3′s properties, such as its high homology to other mitochondrial carriers, especially to UCP2, its short lifetime and low specificity of UCP3 antibodies, have hindered progress in understanding its biological function and transport mechanism over decades. The abundance of UCP3 is highest in murine brown adipose tissue (BAT, 15.0 pmol/mg protein), compared to heart (2.7 pmol/mg protein) and the gastrocnemius muscle (1.7 pmol/mg protein), but it is still 400-fold lower than the abundance of UCP1, a biomarker for BAT. Investigation of UCP3 reconstituted in planar bilayer membranes revealed that it transports protons only when activated by fatty acids (FA). Although purine nucleotides (PN) inhibit UCP3-mediated transport, the molecular mechanism differs from that of UCP1. It remains a conundrum that two homologous proton-transporting proteins exist within the same tissue. Recently, we proposed that UCP3 abundance directly correlates with the degree of FA β-oxidation in cell metabolism. Further development in this field implies that UCP3 may have dual function in transporting substrates, which have yet to be identified, alongside protons. Evaluation of the literature with respect to UCP3 is a complex task because (i) UCP3 features are often extrapolated from its “twin” UCP2 without additional proof, and (ii) the specificity of antibodies against UCP3 used in studies is rarely evaluated. In this review, we primarily focus on recent findings obtained for UCP3 in biological and biomimetic systems.

Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.

We combined recognition imaging and force spectroscopy to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode, a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition images of reconstituted receptors were recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the binding mechanism between mitochondrial uncoupling protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP). Moreover the dynamics of force loading was varied to elucidate the binding dynamics and map the interaction energy landscape.

Plant Photosystem I (PSI) is one of the most intricate membrane complexes in Nature. It is comprised of two complexes, a reaction center and light-harvesting LHCI. An atomic-level structural model of higher plant PSI at 2.2 Å resolution has been constructed based on new crystal form. The structure includes 16 subunits and more than 200 prosthetic groups, the majority of which are light harvesting pigments. The model reveals detailed interactions, providing mechanisms for excitation energy transfer and its modulation in one of Nature's most efficient photochemical machine. Recently we solved the structure of trimeric PSI from Synechocystis at 2.5 Å resolution. Several differences between the mesophilic and thermophilic PSI were revealed and the position of lipids between the monomers was determined. Similarly the structure of monomeric PSI was determined. An operon encoding PSI was identified in cyanobacterial marine viruses. We generated a PSI that mimics the salient features of the viral complex containing PsaJ-F fusion subunit. The mutant is promiscuous for its electron donors and can accept electrons from respiratory cytochromes. We solved the structure of the PsaJ-F fusion mutant as well as a monomeric PSI at 2.8 Å resolution, with subunit composition similar to the viral PSI. The novel structures provided for the first time a detailed description of the reaction center and antenna system from mesophilic cyanobacteria, including red chlorophylls and cofactors of the electron transport chain. Our finding extends the understanding of PSI structure, function and evolution and suggests a unique function for the viral PSI.

resolution has led not only to newly resolved intermediates and pathways, but also to new dynamics between states. For example, the number of detected intermediates in a pair of transmembrane helices increased from 2 to 14, and helix segments were observed to refold against high forces (F=60-140 pN) at standard pulling rates (v=0.1-5 mm/s). True equilibrium measurements characterized the extraction and insertion of small sections of different transmembrane helices. Cooperative folding and unfolding of a 3-amino-acid structural element was resolved in <15 ms at the C-terminal end of helix E. The transition's underlying energy landscape was deduced using a recently developed reconstruction method based on p fold analysis, from which the water-to-bilayer folding free energy of hydrophobic helices were obtained. Similar back-and-forth transitions were observed within a portion of the folding pathway of helix A. These observations provide direct evidence that transmembrane helix segments can dynamically equilibrate between the aqueous and membrane phases, which has been proposed to play important roles in helix cotranslational integration via translocon. Our results demonstrate a new regime in studies of protein-folding dynamics: equilibrium folding and unfolding at 1-ms resolution. Moreover, we applied this technique to the important problem of measuring a membrane protein fold into its native membrane environment.