Amino-acid type identification in 15N-HSQC spectra by combinatorial selective 15N-labelling

Authors

Peter S.C Wu, Australian National University
Kiyoshi Ozawa, University of WollongongFollow
Slobodan Jergic, University of WollongongFollow
Xun Cheng Su, Australian National University
Nicholas E. Dixon, University of WollongongFollow
Gottfried Otting, Australian National University

RIS ID

16017

Publication Details

Wu, P. S.C., Ozawa, K., Jergic, S., Su, X. Cheng., Dixon, N. E. & Otting, G. (2006). Amino-acid type identification in 15N-HSQC spectra by combinatorial selective 15N-labelling. Journal of Biomolecular N M R, 34 (1), 13-21.

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, 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.