Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR.
Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protein by 1H-15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, 15N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using 15N-labeled GB1-peptide fusions and unlabeled target.
Huth
J R
JR
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Bewley
C A
CA
Jackson
B M
BM
Hinnebusch
A G
AG
Clore
G M
GM
Gronenborn
A M
AM
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006609:High Mobility Group Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D017510:Protein Folding; D011494:Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012107:Research Design; D029701:Saccharomyces cerevisiae Proteins; D019014:fas Receptor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006609:High Mobility Group Proteins; C034284:IgG Fc-binding protein, Streptococcus; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019014:fas Receptor; D011494:Protein Kinases