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

Overview

Journal J Biomol NMR

Publisher Springer

Specialties Molecular Biology
Nuclear Medicine

Date 2006 Mar 1

PMID 16505960

Citations 23

Authors

Peter S C Wu

Kiyoshi Ozawa

Slobodan Jergic

Xun-Cheng Su

Nicholas E Dixon

Gottfried Otting

Affiliations

Soon will be listed here.

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 tau subunit of the Escherichia coli DNA polymerase III holoenzyme, tauC16 and tauC14. 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 tauC16 vs. those in tauC14, and the analysis of the structure and mobility of the C-terminal residues present in tauC16 but not in tauC14.

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