期刊
NATURE PROTOCOLS
卷 7, 期 8, 页码 1523-1533出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2012.077
关键词
-
资金
- NIDDK/NIH
We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-'visible' free species and an NMR-'invisible' species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. N-15-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (Delta R-2) in N-15 transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The N-15-DEST and Delta R-2 data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-beta (A beta) at concentrations of similar to 100 mu M.
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