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

Abstract

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, τC16 and τC14. 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 τC16 vs. those in τC14, and the analysis of the structure and mobility of the C-terminal residues present in τC16 but not in τC14. Show more