Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance.

Abstract

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 15N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl 13C atoms experience less isotope scrambling than the main-chain 15N atoms do. However, little is known about the side-chain 13C atoms. Here, the 13C scrambling profiles of the Cα and side-chain carbons were investigated for 15N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in 13Cα and 13C side-chain labeling than in 15N labeling. We utilized this reduced scrambling-prone character of 13C as a simple and efficient method for amino acid selective 13C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the 13C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. 1H-15N heteronuclear single-quantum coherence signals of the 15N scrambling-prone amino acid were also easily filtered using 15N-{13Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.