15N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes

Authors

Kiyoshi Ozawa, University of WollongongFollow
Peter S.C Wu, Australian National University
Nicholas E. Dixon, University of WollongongFollow
Gottfried Otting, Australian National University

RIS ID

16847

Publication Details

Ozawa, K., Wu, P. S.C., Dixon, N. E. & Otting, G. (2006). 15N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes. The FEBS Journal, 273 (18), 4154-4159.

Abstract

Abstract

[¹⁵N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [¹⁵N]-labelled proteins, where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of [¹⁵N]-HSQC spectra by amino acid type selective [¹⁵N]-labelling. The samples can be measured without chromatographic protein purification, dilution of isotopes by transaminase activities are suppressed, and a combinatorial isotope labelling scheme can be adopted that combines reduced spectral overlap with a minimum number of samples for the identification of all [¹⁵N]-HSQC cross-peaks by amino acid residue type. These techniques are particularly powerful for tracking [¹⁵N]-HSQC cross-peaks after titration with unlabelled ligand molecules or macromolecular binding partners. In particular, combinatorial isotope labelling can provide complete cross-peak identification by amino acid type in 24 h, including protein production and NMR measurement.