Precursor-Based Selective Methyl Labeling of Cell-Free Synthesized Proteins

NMR studies of large proteins are complicated by pronounced spectral overlap and large line width. Reducing complexity by [13C, 1H] selective labeling of l-Val, l-Leu, and/or l-Ile residues in combination with optional perdeuteration is therefore commonly approached by supplying labeled amino acid precursors into bacterial expression cultures, although often compromised by high label costs, precursor instability, and label scrambling. Cell-free expression combines efficient production of membrane proteins with significant advantages for protein labeling such as small reaction volumes, defined amino acid pools, and reliable label incorporation. While amino acid specific isotopic labeling of proteins is routine application, the amino acid methyl side-chain labeling was so far difficult as appropriately labeled amino acids are hardly available. On the basis of recent proteome analyses of cell-free lysates, we have developed a competitive strategy for efficient methyl labeling of proteins based on conversion ...