Quantitative Analysis of Protein Backbone Dynamics in Microcrystalline Ubiquitin by Solid-State NMR Spectroscopy
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- 作者：Paul Schanda ; Beat H. Meier ; Matthias Ernst
- 刊名：Journal of the American Chemical Society
- 出版年：2010
- 出版时间：November 17, 2010
- 年：2010
- 卷：132
- 期：45
- 页码：15957-15967
- 全文大小：422K
- 年卷期：v.132,no.45(November 17, 2010)
- ISSN：1520-5126
文摘
Characterization of protein dynamics by solid-state NMR spectroscopy requires robust and accurate measurement protocols, which are not yet fully developed. In this study, we investigate the backbone dynamics of microcrystalline ubiquitin using different approaches. A rotational-echo double-resonance type (REDOR-type) methodology allows one to accurately measure 1H−15N order parameters in highly deuterated samples. We show that the systematic errors in the REDOR experiment are as low as 1% or even less, giving access to accurate data for the amplitudes of backbone mobility. Combining such dipolar-coupling-derived order parameters with autocorrelated and cross-correlated 15N relaxation rates, we are able to quantitate amplitudes and correlation times of backbone dynamics on picosecond and nanosecond time scales in a residue-resolved manner. While the mobility on picosecond time scales appears to have rather uniform amplitude throughout the protein, we unambiguously identify and quantitate nanosecond mobility with order parameters S2 as low as 0.8 in some regions of the protein, where nanosecond dynamics has also been revealed in solution state. The methodology used here, a combination of accurate dipolar-coupling measurements and different relaxation parameters, yields details about dynamics on different time scales and can be applied to solid protein samples such as amyloid fibrils or membrane proteins.