ArticleJournal of biomolecular NMR2003
A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange.
Article in Journal of biomolecular NMR, 2003. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed.
- Side-chain-selective deuterium labeling by a simple bio-expression method enhances resolution and simplifies spectra inJournal of biomolecular NMR · 2025Article
- Protein deuterationChemical communications (Cambridge, England) · 2024Article
- Deuteration of proteins boosted by cell lysates: high-resolution amide and HMagnetic resonance (Gottingen, Germany) · 2024Article
- E. coli "Stablelabel" S30 lysate for optimized cell-free NMR sample preparation.Journal of biomolecular NMR · 2023Article
- Alpha protons as NMR probes in deuterated proteins.Journal of biomolecular NMR · 2019Article
- Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR.Journal of biomolecular NMR · 2015Article
- Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies.Journal of biomolecular NMR · 2009Article
- Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement.Proceedings of the National Academy of Sciences of the United States of America · 2009Article
- Structure of the calcineurin-NFAT complex: defining a T cell activation switch using solution NMR and crystal coordinates.Structure (London, England : 1993) · 2007Article
- 1-13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins.Journal of biomolecular NMR · 2007Article
- Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel.Journal of biomolecular NMR · 2006Article
- NMR study of the tetrameric KcsA potassium channel in detergent micelles.Protein science : a publication of the Protein Society · 2006Article
- Deuterium-resistant algal cell line for D labeling of heterotrophs expresses enhanced level of Hsp60 in D2O medium.Applied and environmental microbiology · 2005Article
- Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background.Journal of biomolecular NMR · 2004Article
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5 authors.
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Abstract
Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30-40 kDa to overcome problems arising from rapid (1)H and (13)C transverse relaxation. However, (1)H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D(2)O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H(2)O. As a side-effect partial C(alpha) protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary (1)H(N)- and (1)H(alpha)-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D(2)O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of C(alpha) protonation of individual amino acids is determined quantitatively and transverse relaxation properties of (1)H(N) and (15)N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.
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