Amino Acid Selective C Labeling and C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance.

Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl C atoms experience less isotope scrambling than the main-chain N atoms do. However, little is known about the side-chain C atoms. Here, the C scrambling profiles of the Cα and side-chain carbons were investigated for N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in Cα and C side-chain labeling than in N labeling. We utilized this reduced scrambling-prone character of C as a simple and efficient method for amino acid selective C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. H-N heteronuclear single-quantum coherence signals of the N scrambling-prone amino acid were also easily filtered using N-{Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.