PMID:12188667
| Citation |
Tugarinov, V, Muhandiram, R, Ayed, A and Kay, LE (2002) Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase g. J. Am. Chem. Soc. 124:10025-35 |
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| Abstract |
A four-dimensional (4-D) NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is described. Virtually complete backbone (1)HN, (15)N, (13)C, and (13)C(beta) chemical shift assignments of this largely alpha-helical protein are reported. The assignment strategy follows from our previously described approach based on TROSY triple resonance 4-D NMR spectroscopy [Yang, D.; Kay, L. E. J. Am. Chem. Soc. 1999, 121, 2571-2575. Konrat, R; Yang, D; Kay, L. E. J. Biomol. NMR 1999, 15, 309-313] with a number of modifications necessitated by the large size of the protein. A protocol for refolding deuterated MSG in vitro was developed to protonate the amides deeply buried in the protein core. Of interest, during the course of the assignment, an isoaspartyl linkage in the protein sequence was unambiguously identified. Chemical shift assignments of this system are a first step in the study of how the domains of the protein change in response to ligand binding and for characterizing the dynamical properties of the enzyme that are likely important for function. |
| Links | |
| Keywords |
Amino Acid Sequence; Escherichia coli/enzymology; Malate Synthase/chemistry; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular/methods; Protein Conformation; Protein Folding; Protein Structure, Secondary; Solutions |
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