Priten Patel

Rutgers University

Schwartz Laboratory
Mentor: James Whittle

Summer 2008

Crystallographic studies of Nucleoporin Nup133

Molecular transport between the nucleus and the cytoplasm is facilitated via the nuclear pore complex (NPC), a 100MDa protein assembly, embedded within the double membrane of the nuclear envelope. The NPC is collectively made up of about 30 proteins termed nucleoporins that compose the structural core of the NPC. Our particular interest is in the middle domain of Nup133, which sits in the central part of the pore, squeezed between the kinked cytoplasmic side and the nuclear side of the NPC. While the structures of the N-and C-termini of Nup133 have been solved, no atomic resolution structure of the middle domain of this protein has been obtained to date. In order to obtain the middle domain, an existing expression plasmid was modified by PCR to encode either residues 517-867 or residues 517-914 of Nup133 as His-tag fusion proteins. These proteins were expressed in E. coli and purified by nickel affinity, ion-exchange, and size exclusion chromatography. Screens for suitable crystallization conditions were carried out using a robotic liquid handling system. An initial condition for crystallization of Nup133 residues 517-914 was identified. The protein crystallized in 1.5M lithium sulfate monohydrate, 0.1 M Bis-tris propane, and at pH 7. Further experiments will be conducted in order to reproduce the crystals.