Protein signal assignments using specific labeling and cell-free synthesis

The goal of structural genomics initiatives is to determine complete sets of protein structures that represent recently sequenced genomes. The development of new high throughput methods is an essential aspect of this enterprise. Residue type and sequential assignments obtained from specifically labeled samples, when combined with 3D heteronuclear data, can significantly increase the efficiency and accuracy of the assignment process, the first step in structure determination by NMR. A protocol for the design of specifically labeled samples with high information content is presented along with a description of the experiments used to extract essential information using 2D versions of 3D heteronuclear experiments. In vitro protein synthesis methods were used to produce four specifically labeled samples of the 23.5 kDa protein phosphoserine phosphatase (PSP) from Methanoccous jannaschii (MJ1594). Each sample contained two (13)C/(15)N-labeled amino acids and one (15)N-labeled amino acid. The 135 type and 14 sequential assignments obtained from these samples were used in conjunction with 3D data obtained from uniformly (13)C/(15)N-labeled and (2)H/(13)C/(15)N-labeled protein to manually assign the backbone (1)H(N), (15)N, (13)CO, (13)C(alpha), and (13)C(beta) signals. Using an automated assignment algorithm, 30% more assignments were obtained when the type and sequential assignments were used in the calculations.