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Quantitative dynamics and binding studies of the 20S proteasome by NMR

Abstract

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.

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Figure 1: Details of the proteasome structure and methyl assignments.
Figure 2: Details of the assignment strategy.
Figure 3: Quantification of dynamics and structure.
Figure 4: Interaction between the 20S proteasome and the 11S activator.

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Acknowledgements

Acknowledgements We thank J. Forman-Kay for discussions and for providing laboratory space, F. Hansen for discussions, J. Rubenstein for electron microscopy images, R. Muhandiram for NMR support and C. Hill for a plasmid of the 11S activator complex. R.S. acknowledges EMBO and the Canadian Institutes of Health Research (CIHR) Training Program in Protein Folding and Disease for fellowships. L.E.K. holds a Canada Research Chair in Biochemistry. Grant support from CIHR and NSERC is acknowledged.

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Correspondence to Lewis E. Kay.

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Supplementary information

Supplementary Information

This file contains Supplementary figures S1-S7, Supplementary Notes which include sample preparation, NMR Experiments and measurement of binding constants and additional references. (PDF 2763 kb)

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Sprangers, R., Kay, L. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445, 618–622 (2007). https://doi.org/10.1038/nature05512

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