Quantitative dynamics and binding studies of the 20S proteasome by NMR.
The machinery used by the cell to perform essential biological processes is made up of large molecular assemblies. One such complex, the proteasome, is the central molecular machine for removal of damaged and misfolded proteins from the cell. Here we show that for the 670-kilodalton 20S proteasome core particle it is possible to overcome the molecular weight limitations that have traditionally hampered quantitative nuclear magnetic resonance (NMR) spectroscopy studies of such large systems. This is achieved by using an isotope labelling scheme where isoleucine, leucine and valine methyls are protonated in an otherwise highly deuterated background in concert with experiments that preserve the lifetimes of the resulting NMR signals. The methodology has been applied to the 20S core particle to reveal functionally important motions and interactions by recording spectra on complexes with molecular weights of up to a megadalton. Our results establish that NMR spectroscopy can provide detailed insight into supra-molecular structures over an order of magnitude larger than those routinely studied using methodology that is generally applicable.
Sprangers
Remco
R
Department of Biochemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Kay
Lewis E
LE
D001419:Bacteria / Q000201:enzymology; D018360:Crystallography, X-Ray; D008745:Methylation; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013822:Thermoplasma / Q000201:enzymology*
D046988:Proteasome Endopeptidase Complex; C119011:26S proteasome non-ATPase regulatory subunit 13