N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes.
[(15)N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [(15)N]-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 [(15)N]-HSQC spectra by amino acid type selective [(15)N]-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 [(15)N]-HSQC cross-peaks by amino acid residue type. These techniques are particularly powerful for tracking [(15)N]-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.
Ozawa
Kiyoshi
K
Research School of Chemistry, Australian National University, Canberra, ACT, Australia.
Wu
Peter S C
PS
Dixon
Nicholas E
NE
Otting
Gottfried
G
D002474:Cell-Free System; D008024:Ligands; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; D009587:Nitrogen Isotopes; D011506:Proteins