15N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexesFaculty of Science, Medicine and Health - Papers
AbstractAbstract [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.
Citation InformationKiyoshi Ozawa, Peter S.C Wu, Nicholas E Dixon and Gottfried Otting. "15N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes" (2006)
Available at: http://works.bepress.com/nick_dixon/54/