Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy.
The development of isotope labeling methodology has had a significant impact on NMR studies of high-molecular-weight proteins and macromolecular complexes. Here we review some of this methodology that has been developed and used in our laboratory. In particular, experimental protocols are described for the production of highly deuterated, uniformly 15N- and 13C-labeled samples of large proteins, with optional incorporation of selective isotope labels into methyl groups of isoleucine, leucine and valine residues. Various types of methyl labeling schemes are assessed, and the utility of different methyl labeling strategies is highlighted for studies ranging from protein structure determination to the investigation of side-chain dynamics. In the case of malate synthase G (MSG), the time frame of the whole preparation, including the protein refolding step, is about 70 h.
Tugarinov
Vitali
V
Department of Medical Genetics and Microbiology, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario, M5S 1A8 Canada.
Kanelis
Voula
V
Kay
Lewis E
LE
D002247:Carbon Isotopes; D003903:Deuterium; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000191:economics / Q000379:methods*; D015394:Molecular Structure; D008970:Molecular Weight; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins
D002247:Carbon Isotopes; D011506:Proteins; D011994:Recombinant Proteins; D003903:Deuterium