Difference: MichaelRout (4 vs. 5)

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Thesaurus Terms: cooperative study, intermolecular interaction, molecular dynamics, proteomics, technology /technique development NIH Roadmap Initiative tag, biotechnology

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: CELLULAR & STRUCTURL BIOLOGY LAB Project Start: 30-SEP-2005 Project End: 31-JUL-2010 ICD: NATIONAL CENTER FOR RESEARCH RESOURCES IRG: ZRG1

Abstract: DESCRIPTION (provided by applicant): Most commercial large-scale production of proteins uses column chromatography and synthetic membranes to fractionate and concentrate proteins. Though the requisite multiple recovery steps increase purity, yields drop quickly and expenses rise. Improving the performance of these processes is therefore a high priority. Nature has already solved this kind of protein enrichment problem with the nuclear pore complex (NPC), the macromolecular machine that efficiently segregates proteins between the nucleus and cytoplasm of all eukaryotic cells. Since we now have an understanding of the mechanism by which the NPC transports proteins, our goal is to mimic the molecular machinery of the NPC, with its exquisite selectivity and high throughput, in a robust synthetic platform. Our approach is one of reverse engineering, in which we will dissect the nuclear transport system to elucidate its key elements in order to duplicate them. We will use the yeast NPC, the best understood system, as our starting point. Our approach is one of reverse engineering, in which we will dissect the nuclear transport system to elucidate its key elements in order to duplicate them. We will use the yeast NPC, the best understood system, as our starting point. Specific Aim 1 and Specific Aim 2 seek to detail how the NPC is configured to function as a transporter. For this, we must separate those components that are essential for transport from those needed for other NPC functions, such as self-assembly and NE maintenance, and learn how those components function. Following this, we will study in detail the behavior of the key components needed for nuclear transport, to understand why they make the NPC function so efficiently in vivo. In Specific Aim 3 we will develop a computational simulation of the NPC to explore how to translate these optimal parameters into an artificial machine, and in Specific Aim 4 we will explore several avenues to build such machines at various scales.

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Thesaurus Terms: artificial membrane, bioengineering /biomedical engineering, nuclear membrane, protein purification active transport, mathematical model, protein transport computer simulation, nanotechnology, yeast

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2007 Department: CELLULAR & STRUCTURL BIOLOGY LAB Project Start: 15-SEP-2004 Project End: 31-AUG-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1

Abstract: DESCRIPTION (provided by applicant): ABSTRACT Nuclear pore complexes (NPCs) are the sole mediators of exchange across the nuclear envelope (NE) between the nuclear and cytoplasmic compartments. Nucleocytoplasmic transport depends on the interplay between transport cargoes, their cognate soluble transport factors (many termed Kaps), and NPCs. We have taken a comprehensive approach to defining the functional architecture of the NPC in the model eukaryote Saccharomyces (yeast). We identified all the yeast NPC proteins (Nups) and plotted their disposition in the NPC; this work allowed us to propose a new "virtual gating" mechanism for nuclear transport. We also assigned fold types to all the Nups and systematically isolated Nup subcomplexes to determine the network of interactions they make. We then used this information to compute a 3D map of the NPC architecture, sufficient to resolve the molecular organization of the entire NPC. Our work exposed a simple modularity in the architecture of the NPC; moreover, similarities between structures in coated vesicles and those in the NPC suggest their common evolutionary origin in a progenitor "protocoatomer". Our goal is now to produce high resolution dynamic maps of the NPC. First, we will use additional "low-fruit, high-payoff' immunopurification and immunolocalization experiments to rapidly improve our NPC map, to the point at which we can discern the shapes of the Nups in it. We will then study recombinant Nups and Nup complexes using electron microscopy and crosslinking, to reveal fine details on how the folds, domains, and proteins are organized within the Nups and their complexes. Next, we will reconstitute key reactions of nucleocytoplasmic transport in vitro, and test possible mechanistic models in vivo, in order to reconstruct the movements Kaps and their cargos make on crossing the NPC. We will finally synergistically decode this information and convert it into dynamic, 3D representations of the NPC and nuclear transport, ideally at atomic resolution, thereby allowing us to understand the origin, assembly and mechanism of the NPC at the most fundamental level. LAY SUMMARY We are studying the tiny machines that shuttle materials to the DNA in living cells. These machines, called "nuclear pore complexes", allow the DNA to send its instructions to the rest of the cell, and so help regulate how a cell lives, develops, and stops itself from making the kinds of mistakes seen in cancer cells. We wish to understand how these machines work and how they arose in the early evolution of life.

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Thesaurus Terms: cell nucleus, intracellular transport, membrane activity, membrane transport protein, nuclear membrane, structural biology G protein, binding protein, biological model, biophysics, cell component structure /function, cytoplasm, intermolecular interaction, molecular assembly /self assembly, molecular asymmetry, molecular site, protein protein interaction, transport protein affinity chromatography, affinity labeling, mass spectrometry, protein purification, reporter gene, yeast

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: CELLULAR & STRUCTURL BIOLOGY LAB Project Start: 01-JAN-2001 Project End: 30-JUN-2011 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: NDT