We seek to provide structural insights into thermoregulation, energy metabolism and the maintenance of normal mitochondrial membrane potentials in mammals. The portion of mitochondria! H+ electrochemical potential that is dissipated through pathways other than ADP-ATP conversion is termed """"""""uncoupling"""""""" and is primarily mediated by the H+ translocation activity of uncoupling proteins (UCPs). UCPs are ~32 kD integral membrane proteins of the mitochondrial inner membrane, whose functions are regulated by purine nucleotides and fatty acids (FAs). UCPs are important therapeutic targets. For example, UCPs activators could promote a negative cell energy balance, for the treatment of obesity or UCP2 inhibitors could restore pancreatic b-cell glucose sensing, for the treatment of type-2 diabetes. Despite the strong interest in these proteins, little is known about the mechanism of H+ translocation and regulation due partly to the lack of structural information. We propose detailed structural studies on UCP1 and UCP2 at various physiological states. The proposal consists of two aims. First, we will determine, by solution NMR, the structure of the GDP-inhibited UCP2. A NMR approach to be explored and developed for this goal is the use of orientation restraints in the form of dipolar couplings and distance restraints from paramagnetic spin labels to obtain high-resolution structure with minimal amount of NMR spectral analysis. Dynamic properties of UCP2 will be characterized by NMR relaxation measurements. We will also screen for crystals of UCP1 and UCP2 for obtaining x-ray structures of their inhibited states. The structure and dynamic properties from NMR studies will be used to guide deletions of flexible regions not required for function, or for the design of disulfide bridges to improve conformational homogeneity and facilitate crystallization. Second, we will determine the structure of the FA-activated UCP2 by NMR using protocols established in aim 1, as well as carry out crystallographic studies on the active forms of UCP1 and UCP2. Structure and dynamics of the active and inhibited forms will be compared to elucidate a mechanism of H+ translocation. Overall, this is a proof-of- principle study aimed to establish NMR and crystallization protocols for structural studies of UCP1 and/or UCP2, such that working protocols can be employed in the future for structural studies of other members of the UCP family for acquiring a thorough mechanistic understanding of the activities of these proteins. ? ? ?
|Berardi, Marcelo J; Shih, William M; Harrison, Stephen C et al. (2011) Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching. Nature 476:109-13|