Amino-acid Type Identification in super(15)N-HSQC Spectra by Combinatorial Selective super(15)N-labelling

The efficiency of cell-free protein synthesis combined with combinatorial selective super(15)N-labelling provides a method for the rapid assignment of super(15)N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five super(15)N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the tau subunit of the Escherichia coli DNA polymerase III holoenzyme, tau sub(C)16 and tau sub(C)14. Since each of the five super(15)N-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled samples, spectral overlap was much reduced. All super(15)N-HSQC cross-peaks of the backbone amides could be assigned to the correct amino-acid type. Availability of the residue-type information greatly assisted the evaluation of the changes in chemical shifts observed for corresponding residues in tau sub(C)16 vs. those in tau sub(C)14, and the analysis of the structure and mobility of the C-terminal residues present in tau sub(C)16 but not in tau sub(C)14.