Expression, Reconstitution and Biophysical Studies of Neuronal Uncoupling Proteins: UCP4 and UCP5

Abstract

Uncoupling proteins (UCPs), located in the inner membrane of the mitochondria, uncouple ATP-synthesis from the respiratory chain by transporting protons across the inner membrane into the matrix, hence dissipating the proton-motive force and releasing heat. The neuronal UCPs (nUCPs), UCP4 and UCP5, were discovered recently (in 1998) and little is known about their structure and function. To gain further insight into the potential importance of these two proteins in the neuroprotection and neuromodulation of neurodegenerative diseases, this study will focus on the structure, function and interaction of the nUCPs with nucleotides (inhibitors) and fatty acids (activators). A recombinant version of the proteins, utilizing a hexa-histidine tag and a TEV protease site (for subsequent His-tag cleavage) has been designed, expressed as insoluble inclusion bodies, and isolated and purified using immobilized metal affinity chromatography. Subsequent reconstitution of the proteins in mild detergent (DDM and digitonin) allowed for biophysical studies by circular dichroism and fluorescence spectroscopy. Circular dichroism spectroscopy has shown that, similar to the recombinant UCP1, nUCPs possess dominantly helical structures in digitonin and DDM [1]. Furthermore, detergent-mediated reconstitution of the proteins into preformed liposomes can give more physiologically relevant structural and functional information. Comparison of the structure and function of human UCP1 (thermogenin) to nUCPs, in lipid membranes and membrane-like environments, will eventually show whether these proteins have any similarity in conformation and functional behaviour.[1] Jelokhani-Niaraki, M., Ivanova, M.V., McIntyre, B.L., Newman, C.L., McSorley, F.R., Young, E.K. and Smith, M.D. (2008) Biochem. J., 411, 593-603.