Protein labeling strategies for liquid-state NMR spectroscopy using cell-free synthesis

摘要

Preparation of a protein sample for liquid-state nuclear magnetic resonance (NMR) spectroscopy analysis requires optimization of many parameters. This review describes labeling strategies for obtaining assignments of protein resonances. Particular emphasis is placed on the advantages of cell-free protein production, which enables exclusive labeling of the protein of interest, thereby simplifying downstream processing steps and increasing the availability of different labeling strategies for a target protein. Furthermore, proteins can be synthesized in milligram yields, and the open nature of the cell-free system allows the addition of stabilizers, scrambling inhibitors or hydrophobic solubilization environments directly during the protein synthesis, which is especially beneficial for membrane proteins. Selective amino acid labeling of the protein of interest, the possibility of addressing scrambling issues and avoiding the need for labile amino acid precursors have been key factors in enabling the introduction of new assignment strategies based on different labeling schemes as well as on new pulse sequences. Combinatorial selective labeling methods have been developed to reduce the number of protein samples necessary to achieve a complete backbone assignment. Furthermore, selective labeling helps to decrease spectral overlap and overcome size limitations for solution NMR analysis of larger complexes, oligomers, intrinsically disordered proteins and membrane proteins. (C) 2017 Elsevier B.V. All rights reserved.