The nuclear pore complex (NPC) is one of the most important molecular machines in eukaryotes because it gates the porous conduits between the cytoplasm and nucleoplasm of cells and controls all nucleo-cytoplasmic traffic and communication. Its most important architectural feature is a poorly understood semi-permeable diffusion barrier in its center that maintains a tight seal against cytoplasmic proteins as small as 4 nanometers in size, but opens to allow facilitated transport of particles of all shapes and sizes up to 40 nanometers in size. This flexible barrier is composed of a family of filamentous proteins named FG nucleoporins (FG nups) that feature large unfolded domains in their native functional state, which are decorated with multiple phenylalanine glycine motifs (FG domains).
The specific aims of our proposed research are to 1) characterize the dynamic structure and intra-molecular interactions of FG domains representing two different types of FG nup filaments that are anchored at three different locales of the NPC, and 2) test the hypothesis that inter-molecular associations between FG domains of nups create a filamentous meshwork structure at the NPC center, which establishes the size-selective barrier to the passive diffusion of proteins. The proposed experiments will combine biochemical, biophysical, cell biological, structural (Nuclear Magnetic Resonance), and molecular modeling techniques to gain insight into the dynamic behavior, structure and function of the FG nups. We will also gain fundamental knowledge on the dynamic behavior and structure of disordered domains of proteins in general. We are studying the three-dimensional structure of the cellular proteins that function as gatekeepers of our genetic material in the nucleus. Their proper architecture and function is vital to human health because they control the flow of information to and from our genes. When they fail to function normally, these proteins can trigger the onset of cancer. We wish to understand how their structure enables them to function as gatekeepers of the nucleus.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077520-03
Application #
7683744
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Shapiro, Bert I
Project Start
2007-09-28
Project End
2011-08-31
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
3
Fiscal Year
2009
Total Cost
$333,083
Indirect Cost
Name
University of California Santa Cruz
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
125084723
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
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Heisel, Kurt A; Krishnan, V V (2014) NMR based solvent exchange experiments to understand the conformational preference of intrinsically disordered proteins using FG-nucleoporin peptide as a model. Biopolymers 102:69-77
Rangl, Martina; Ebner, Andreas; Yamada, Justin et al. (2013) Single-molecule analysis of the recognition forces underlying nucleo-cytoplasmic transport. Angew Chem Int Ed Engl 52:10356-9
Ando, David; Colvin, Michael; Rexach, Michael et al. (2013) Physical motif clustering within intrinsically disordered nucleoporin sequences reveals universal functional features. PLoS One 8:e73831
Phillips, Joshua L; Colvin, Michael E; Newsam, Shawn (2011) Validating clustering of molecular dynamics simulations using polymer models. BMC Bioinformatics 12:445
Yamada, Justin; Phillips, Joshua L; Patel, Samir et al. (2010) A bimodal distribution of two distinct categories of intrinsically disordered structures with separate functions in FG nucleoporins. Mol Cell Proteomics 9:2205-24
Mielke, Steven P; Krishnan, V V (2009) Characterization of protein secondary structure from NMR chemical shifts. Prog Nucl Magn Reson Spectrosc 54:141-165
Krishnan, V V; Lau, Edmond Y; Yamada, Justin et al. (2008) Intramolecular cohesion of coils mediated by phenylalanine--glycine motifs in the natively unfolded domain of a nucleoporin. PLoS Comput Biol 4:e1000145