Multidimensional NMR spectroscopy for protein characterization and assignment inside cells.
High-field, heteronuclear NMR spectroscopy of biological macromolecules in native cellular environments is limited by the low concentrations present and the long data acquisition times needed for the experiments. Successful 1D and 2D heteronuclear NMR data have been reported, but the 3D experiments conventionally used for protein assignment and detailed characterization are generally too long to maintain cell viability. Here we describe the successful in vivo implementation of a suite of fast 3D NMR experiments which we have used to generate the complete backbone assignment of resonances in the recombinant polypeptide GB-1 within Escherichia coli cells. The data were acquired at 600 MHz with a cold probe using the projection reconstruction experiments, (3,2)HNCA, (3,2)HNCO, and (3,2)HA(CA)NH.
Reardon
Patrick N
PN
Departments of Biochemistry and Radiology, Duke University Medical Center, Durham, NC 27710, USA.
Spicer
Leonard D
LD
D004591:Electrophoresis, Polyacrylamide Gel; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
D011506:Proteins