AN AUTOMATED PROCEDURE FOR THE ASSIGNMENT OF PROTEIN (HN)-H-1,N-15, C-13(ALPHA), H-1(ALPHA), C-13(BETA) AND H-1(BETA) RESONANCES

A computer algorithm that determines the (HN)-H-1, N-15, C-13(alpha), H-1(alpha) C-13(beta) and H-1(beta) chemical-shift assignments of prot ein residues with minimal human intervention is described. The algorit hm is implemented as a suite of macros that run under a modified versi on of the FELIX 1.0 program (Hare Research, Bothell, WA). The input to the algorithm is obtained from six multidimensional, triple-resonance experiments: 3D HNCACB, 3D CBCA(CO)HN, 4D HNCAHA, 4D HN(CO)CAHA, 3D H BHA(CO)NH and 3D HNHA(Gly). For small proteins, the two 4D spectra can be replaced by either the 3D HN(CA)HA, 3D H(CA)NNH, or the N-15-edite d TOCSY-HSQC experiments. The algorithm begins by identifying and coll ecting the intraresidue and sequential resonances of the backbone and C-13(beta) atoms into groups. These groups are sequentially linked and then assigned to residues by matching the C-13(alpha) and C-13(beta) chemical-shift profiles of the linked groups to that of the protein's primary structure. A major strength of the algorithm is its ability to overcome imperfect data, e.g., missing or overlapping peaks. The viab ility of the procedure is demonstrated with two test cases. In the fir st, NMR data from the six experiments listed above were used to reassi gn the backbone resonances of the 93-residue human hnRNP C RNA-binding domain. In the second, a simulated cross-peak list, generated from th e published NMR assignments of calmodulin, was used to test the abilit y of the algorithm to assign the backbone resonances of proteins conta ining internally homologous segments. Finally, the automated method wa s used to assign the backbone resonances of apokedarcidin, a previousl y unassigned, 114-residue protein.