Structure of Nickel/Cobalt Permeases (NiCoT; TC 2.A.52)

Nickel/cobalt transporters (NiCoT, TC 2.A.52) mediate the high-affinity uptake of Ni2+ and Co2+ ions in prokaryotes and certain fungi. Relatives with significant sequence similarity to these systems are widespread in plants. It is the goal of this project to characterize the molecular basis of selective metal binding and transport of NiCoTs using crystallographic and biophysical techniques. In the first application period, protocols for the large-scale purification of NiCoTs have been established and 2D and 3D crystallization approaches have been initiated. The focus of the forthcoming period will be on optimization of the crystallization conditions (i) aiming at the generation of crystals that are suitable for X-ray diffraction studies and (ii) yielding a 3D structural model at a reasonable resolution. Site-directed mutagenesis and various spectroscopic techniques will be applied to characterize the metal-liganding sphere in NiCoTs. Similar approaches will be followed to analyze recently recognized putative Ni2+ and Co2+ transporters from the outer membrane of certain gram-negative bacteria.