| Autor: |
Jonathan M, Lamley, Dinu, Iuga, Carl, Öster, Hans-Juergen, Sass, Marco, Rogowski, Andres, Oss, Jaan, Past, Andres, Reinhold, Stephan, Grzesiek, Ago, Samoson, Józef R, Lewandowski |
| Rok vydání: |
2014 |
| Předmět: |
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| Zdroj: |
Journal of the American Chemical Society. 136(48) |
| ISSN: |
1520-5126 |
| Popis: |
NMR spectroscopy is a prime technique for characterizing atomic-resolution structures and dynamics of biomolecular complexes but for such systems faces challenges of sensitivity and spectral resolution. We demonstrate that the application of (1)H-detected experiments at magic-angle spinning frequencies of50 kHz enables the recording, in a matter of minutes to hours, of solid-state NMR spectra suitable for quantitative analysis of protein complexes present in quantities as small as a few nanomoles (tens of micrograms for the observed component). This approach enables direct structure determination and quantitative dynamics measurements in domains of protein complexes with masses of hundreds of kilodaltons. Protein-protein interaction interfaces can be mapped out by comparison of the chemical shifts of proteins within solid-state complexes with those of the same constituent proteins free in solution. We employed this methodology to characterize a300 kDa complex of GB1 with full-length human immunoglobulin, where we found that sample preparation by simple precipitation yields spectra of exceptional quality, a feature that is likely to be shared with some other precipitating complexes. Finally, we investigated extensions of our methodology to spinning frequencies of up to 100 kHz. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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