| 研究実績の概要 |
Proteins that are large, membrane-bound, or studied in living cells by in-cell NMR can in general not be assigned by the conventional solution NMR method that relies on uniform 13C/15N-labeling because the resonance lines become too broad and overlapping. Sparse labeling, in particular of methyl groups is an approach to restore interpretable spectra by drastically reducing relaxation rates and the number of signals. However, it remains difficult to establish resonance assignments, even if the three-dimensional structure of the protein is already known. In this project we develop computational methods to assign the spectra of sparsely labeled difficult proteins by structure-based automated assignment. On the basis of our existing FLYA algorithm for the assignment of uniformly labeled proteins, a new automated assignment method has been developed that can assign large proteins using methyl group labeling or other sparse labeling techniques, and NOESY spectra in conjunction with a known three-dimensional structure. The FLYA algorithm finds assignments by optimizing a mapping between the expected peaks, which one anticipates to see based on the protein sequence, and the measured peaks, which have been identified by peak picking. The general design of the algorithm makes it possible to exploit peak lists from virtually any type of NMR spectrum. In particular, we could show that FLYA is able to assign proteins using as input exclusively NOESY spectra. The new approach has been applied to large proteins up to 468 kDa size and to proteins in living cells.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
A full account of our methyl-FLYA method for structure-based assignment of large, specifically methyl-labeled proteins has been submitted to Nature Communications and is currently under revision. The method has been applied to the automated assignment of five proteins with sizes between 27 and 360 kDa, for which experimental NOESY spectra are available.
The first structure determinations of proteins directly in living eukaryotic cells, which use the automated assignment method, have been completed and published in Angewandte Chemie International Edition.
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