Determining the Molecular Structure of the N-terminal Domain of Nup358 of the Cytoplasmic Filaments of the Nuclear Pore Complex

Reeti K. Gulati, California Institute of Technology

Abstract

The Hoelz Lab studies the nuclear pore complex (NPC), a large macromolecular transport channel imbedded in the membrane of the nucleus that conducts bidirectional transport of cargos such as mRNA. The NPC is essential for the proper function of a eukaryotic cell and thus its defects have been connected to numerous diseases including leukemia, hematological neoplasms, heart arrhythmia, and acute necrotizing encephalopathy. This research was specifically conducted on the nucleoporin Nup358 which is a part of the NPC’s cytoplasmic filaments. This project’s aim was to verify or reject a hypothesized model of the composite structure of Nup358’s N-terminal domain which was derived from the individual structures of residues 1-145 and 145-752 of the protein via x-ray crystallography. To determine the structure of the complete N-terminal domain, we designed, expressed, and purified various constructs that begin in the range of residues 1-145 and end at 752. Thus, the constructs that we expressed overlapped both of the previous constructs whose structures have already been determined. Cloning was conducted to create the specific DNA constructs desired. Then, they were expressed and purified thoroughly via His affinity, buffer exchange, ion exchange, and gel filtration. Crystallization of the proteins was achieved via hanging drop vapor diffusion. Crystallization was optimized by setting up various conditions with different reagents. X-ray diffraction experiments were conducted and a resolution of approximately 7 Angstroms was obtained. The data was used to solve the structure of construct 128-752 which did not appear as predicted due to lack of the additional alpha helix so further investigation will be conducted.

 
Nov 12th, 11:15 AM Nov 12th, 11:30 AM

Determining the Molecular Structure of the N-terminal Domain of Nup358 of the Cytoplasmic Filaments of the Nuclear Pore Complex

HUB 379

The Hoelz Lab studies the nuclear pore complex (NPC), a large macromolecular transport channel imbedded in the membrane of the nucleus that conducts bidirectional transport of cargos such as mRNA. The NPC is essential for the proper function of a eukaryotic cell and thus its defects have been connected to numerous diseases including leukemia, hematological neoplasms, heart arrhythmia, and acute necrotizing encephalopathy. This research was specifically conducted on the nucleoporin Nup358 which is a part of the NPC’s cytoplasmic filaments. This project’s aim was to verify or reject a hypothesized model of the composite structure of Nup358’s N-terminal domain which was derived from the individual structures of residues 1-145 and 145-752 of the protein via x-ray crystallography. To determine the structure of the complete N-terminal domain, we designed, expressed, and purified various constructs that begin in the range of residues 1-145 and end at 752. Thus, the constructs that we expressed overlapped both of the previous constructs whose structures have already been determined. Cloning was conducted to create the specific DNA constructs desired. Then, they were expressed and purified thoroughly via His affinity, buffer exchange, ion exchange, and gel filtration. Crystallization of the proteins was achieved via hanging drop vapor diffusion. Crystallization was optimized by setting up various conditions with different reagents. X-ray diffraction experiments were conducted and a resolution of approximately 7 Angstroms was obtained. The data was used to solve the structure of construct 128-752 which did not appear as predicted due to lack of the additional alpha helix so further investigation will be conducted.