Segmental isotopic labeling of proteins for nuclear magnetic resonance.
Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the three-dimensional structures under near physiological conditions but also a convenient method for studying protein-ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation, caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental isotopic labeling allows for specific segment(s) within a protein to be selectively examined by NMR, thus significantly reducing the spectral complexity for large proteins and allowing for the application of a variety of solution-based NMR strategies. Two related approaches are generally used in the segmental isotopic labeling of proteins: expressed protein ligation and protein trans-splicing. Here, we describe the methodology and recent application of expressed protein ligation and protein trans-splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental isotopic labeling of a 50-kDa protein Csk (C-terminal Src kinase) using expressed protein ligation methods.
Liu
Dongsheng
D
New York Structural Biology Center, New York, USA.
Xu
Rong
R
Cowburn
David
D
D000595:Amino Acid Sequence; D053000:Analytic Sample Preparation Methods / Q000379:methods*; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D047668:Inteins / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D015080:Mesna; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D019061:src-Family Kinases
D009587:Nitrogen Isotopes; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D015080:Mesna