Site-specific isotopic labeling of proteins for NMR studies
- Univ. of California, Berkeley, CA (United States); and others
NMR spectroscopy is a sensitive, site-specific probe of biomolecular structure. For relatively small proteins and peptides, the H resonances can be assigned using the sequential method. However, there are many cases, especially larger proteins, in which the spectra are too complex for complete, systematic resonance assignments. In some cases, assignments can be made by selective isotopic labeling (e.g., uniform incorporation of a {sup 13}C-labeled amino acid) in conjunction with site-directed mutagenesis or {sup 13}C, {sup 15}N double labeling of adjacent amino acids. However, in many large proteins, protein complexes, and unfolded proteins, resonance overlap and broadening prevents assignments. The ability to synthesize proteins with unnatural amono acids, beyond those specified by the genetic code, makes it possible to isotopically label a single amino acid residue in a protein. We report here the use of this approach to generate a T4 lysozyme (T4L) mutant containing a unique {sup 13}C-labeled alanine, for which {sup 13}C-filtered proton spectra were obtained in both the native and denatured states. This general methodology should be applicable to a variety of NMR measurements in large proteins and protein complexes. 14 refs., 1 fig.
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC03-76SF00098
- OSTI ID:
- 567318
- Journal Information:
- Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 20 Vol. 114; ISSN JACSAT; ISSN 0002-7863
- Country of Publication:
- United States
- Language:
- English
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