Uniform and Residue-specific 15N-labeling of Proteins on a Highly Deuterated Background |
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Authors: | Jocelyne Fiaux Eric B Bertelsen Arthur L Horwich Kurt Wüthrich |
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Institution: | 1. Eidgen?ssische Technische Hochschule Zürich, Institut für Molekularbiologie und Biophysik, CH–8093, Zürich, Switzerland 2. Howard Hughes Medical Institute and Department of Genetics, Yale School of Medicine, New Haven, CT, 06510, U.S.A
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Abstract: | A general method for stable-isotope labeling of large proteins is introduced and applied for studies of the E. coli GroE chaperone proteins by solution NMR. In addition to enabling the residue-specific (15)N-labeling of proteins on a highly deuterated background, it is also an efficient approach for uniform labeling. The method meets the requirements of high-level deuteration, minimal cross-labeling and high protein yield, which are crucial for NMR studies of structures with sizes above 150 kDa. The results obtained with the new protocol are compared to other strategies for protein labeling, and evaluated with regard to the influence of external factors on the resulting isotope labeling patterns. Applications with the GroE system show that these strategies are efficient tools for studies of structure, dynamics and intermolecular interactions in large supramolecular complexes, when combined with TROSY- and CRINEPT-based experimental NMR schemes. |
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Keywords: | large molecular structures residue-specific isotope labeling solution NMR stable-isotope labeling uniform deuterium labeling |
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