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 N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. CarbonylC atoms experience less isotope scrambling than the main-chain N atoms do. However, little is known about the side-chainC atoms. Here, the C scrambling profiles of the Cα and side-chain carbons were investigated forN scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in Cα andC side-chain labeling than in N labeling. We utilized this reduced scrambling-prone character ofC 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, theC 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 usingN-{Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

Segmental and site-specific isotope labelling strategies for structural analysis of posttranslationally modified proteins

“…42 However, metabolic labelling processes used for residue specific labelling might cause potential scrambling of isotope labels due to metabolic interconversion of the amino acids. 43,44 Labelling of single amino acid residues is a potential strategy when focusing on one or very few key positions in a protein. This strategy allows very simplified signal assignments and easy monitoring of reactions involving the labelled key residue(s), but comes with the disadvantage of ignoring the larger protein context (Figure 1c).…”

Efficient production of a functional G protein-coupled receptor in E. coli for structural studies

“…for V172, V202, and V314 (Supplementary Figure S2). However, due to the metabolic scrambling of 15 N-valine to alanine, isoleucine, glutamate and other branched amino acids (Lacabanne et al 2018;Sugiki et al 2018 weakened (e.g. V95, which is already weak in the insect cell spectrum, is not even detected in the E. coli spectrum).…”