International Research Fellowship Program: Characterizing the Proteolytic Chamber of the Proteasome Using Solution NMR

Hansen Alexandar L, Ypsilanti MI

Investigators

Abstract

0852964 Hansen This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). The International Research Fellowship Program enables U.S. scientists and engineers to conduct nine to twenty-four months of research abroad. The program's awards provide opportunities for joint research, and the use of unique or complementary facilities, expertise and experimental conditions abroad. This award will support a twenty-four month research fellowship by Dr. Alexander L. Hansen to work with Dr. Lewis E. Kay at the University of Toronto in Canada. Large biomolecular complexes, such as the proteasome, are common amongst the machinery that makes up a cell. Gaining insight into the function of these large molecular machines often requires detailed information regarding the motions and molecular interactions that are crucial for their biological functions. Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful tool capable of studying the structure and dynamics of biomolecules with atomic resolution. Building on previous work using methyl groups as probes for the molecular dynamics of supramolecular complexes, the target of this study is the 20S proteasome from the Archaeon T. acidophilum, a 670 kDa molecular machine with a focus on its ?Ò-ring proteolytic chamber, the dynamics of this region, and interactions between the proteasome and substrates. There are many fundamental questions that remain to be answered and that can only be addressed via high resolution solution studies. These include understanding the basic mechanism by which product is extruded, the mode of interaction of targets with the proteasome, including unfolded polypeptide chains, and structures of substrates within the antechambers of the proteasome prior to their translocation into the proteolytic chamber. The molecular weights of proteasome complexes can be in excess of one megadalton and new technology and strategies must be developed to answer these fundamental questions. Recently, new methodologies have been developed which utilize the unique spectral properties of methyl groups to address some of these questions. The major goals of this proposal are to (a) develop further the NMR technologies necessary for the study of supra-molecular structures over a large range of timescales and (b) obtain important biological insight into the function of the proteolytic core of the proteasome. This study is providing crucial insight into the biochemistry of the proteasome that builds upon previous structural biology by going beyond static three-dimensional structures to watch these machines in motion to monitor their dynamics which are critical for function. The approach facilitates the study of complexes comprised of components with binding constants sufficiently low, or are partially unfolded, that they cannot be investigated by other structural techniques such as X-ray or cryo-EM. It is still the case that NMR applications are limited to rather small proteins and protein complexes. Since specific cellular tasks are often performed by machinery made up of large molecular assemblies, the methodologies being developed to study such systems are critically important. Given the unique ability of NMR to provide structural and dynamic information with atomic resolution over a range of biologically relevant timescales, the methodologies developed to study the proteasome push NMR technology to new frontiers of discovery.