Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background.
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.
Fiaux
Jocelyne
J
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, CH-8093 Zürich, Switzerland.
Bertelsen
Eric B
EB
Horwich
Arthur L
AL
Wüthrich
Kurt
K
D018835:Chaperonin 10 / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009584:Nitrogen / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012984:Software; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry
D018835:Chaperonin 10; D018834:Chaperonin 60; D003432:Cross-Linking Reagents; D007554:Isotopes; D011506:Proteins; D011994:Recombinant Proteins; D003903:Deuterium; D013762:Tetrahydrofolate Dehydrogenase; D009584:Nitrogen