A COMPUTER-BASED PROTOCOL FOR SEMIAUTOMATED ASSIGNMENTS AND 3D STRUCTURE DETERMINATION OF PROTEINS

A strategy is presented for the semiautomated assignment and 3D struct ure determination of proteins from heteronuclear multidimensional Nucl ear Magnetic Resonance (NMR) data. This approach involves the computer -based assignment of the NMR signals, identification of distance restr aints from nuclear Overhauser effects, and generation of 3D structures by using the NMR-derived restraints. The protocol is described in det ail and illustrated on a resonance assignment and structure determinat ion of the FK506 binding protein (FKBP, 107 amino acids) complexed to the immunosuppressant, ascomycin. The 3D structures produced from this automated protocol attained backbone and heavy atom rmsd of 1.17 and 1.69 Angstrom, respectively. Although more highly resolved structures of the complex have been obtained by standard interpretation of NMR da ta (Meadows et al. (1993) Biochemistry, 32, 754-765), the structures g enerated with this automated protocol required minimal manual interven tion during the spectral assignment and 3D structure calculations stag es. Thus, the protocol may yield an approximate order of magnitude red uction in the time required for the generation of 3D structures of pro teins from NMR data.