*Article* **Molecular Dynamics Simulations of Mitochondrial Uncoupling Protein 2**

**Sanja Škulj 1, Zlatko Brkljaˇca 1, Jürgen Kreiter 2, Elena E. Pohl 2,\* and Mario Vazdar 1,3,\***


**Abstract:** Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreemen<sup>t</sup> with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further sugges<sup>t</sup> that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.

**Keywords:** membrane protein; long-chain fatty acid; proton transfer; purine nucleotide; conductance measurements in model membranes; uncoupling
