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

The efficiency of cell-free protein synthesis combined with combinatorial selective ¹⁵N-labelling provides a method for the rapid assignment of ¹⁵N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five ¹⁵N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the τ subunit of the Escherichia coli DNA polymerase III holoenzyme, τ_C16 and τ_C14. Since each of the five ¹⁵N-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled samples, spectral overlap was much reduced. All ¹⁵N-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 τ_C16 vs. those in τ_C14, and the analysis of the structure and mobility of the C-terminal residues present in τ_C16 but not in τ_C14.