Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

Abstract

Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1–5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-012-9628-3) contains supplementary material, which is available to authorized users.

Segmental isotopic labeling of a single‐domain globular protein without any refolding step by an asparaginyl endopeptidase

“…EPL, PTS, and SrtA have been successfully used for segmental isotopic labeling of multidomain proteins containing self-contained domains [5,6,[32][33][34]. EPL, PTS, and SrtA have been successfully used for segmental isotopic labeling of multidomain proteins containing self-contained domains [5,6,[32][33][34].…”

“…Ligation of protein fragments with peptide bonds could also facilitate production of toxic proteins in vitro. 1B) [5][6][7][8][9][10][11]. Ligation of a synthetic unprotected peptide or a recombinant protein/peptide with a-thio-ester and a peptide bearing an N-terminal cysteine has been achieved by native chemical ligation (NCL; Fig.…”

“…Protein ligation by protein trans-splicing (PTS) via split inteins or split HINT domains is not restricted by Cys residue at the ligation junction ( Fig. 1B) [5][6][7][8][9][10][11]. However, the target protein sequences have to be genetically fused with split intein fragments and the split protein fragments have to be reconstituted into an active conformation for protein ligation.…”

Structural basis for protein trans‐splicing by a bacterial intein‐like domain – protein ligation without nucleophilic side chains

“…The NMR experiments were recorded at 298 K with a 1.2 mM protein solution in 20 mM sodium phosphate (pH 6.0). For the resonance assignments, a set of two-dimensional and 3D spectra were recorded: [ 1 H, 15 For structure determination, a 15 N-edited NOESY-HSQC and aliphatic 13 C-edited NOESY-HSQC spectra, both with a mixing time of 80 ms, were recorded at 1 H frequency of 800 MHz. T 1 and T 2 relaxation and heteronuclear 15 N{ 1 H}-NOEs measurements were performed at 1 H frequency of 600 MHz [39].…”

Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology

“…Different methods, known as native chemical ligation (NCL), expressed protein ligation (EPL), and protein trans-splicing (PTS), are available for segmental isotope labeling of proteins (David et al 2004;Muralidharan and Muir 2006). To date, isotope segmental labeling has not been extensively applied in NMR, although different studies have already demonstrated that segmental labeling is a very elegant and relevant approach to investigate large proteins (Yagi et al 2004;Minato et al 2012), to study conformational changes and ligand binding (Anderson et al 2005), to investigate inter domain interactions within multi-domain proteins (Camarero et al 2002;Zhang et al 2007), and also to enable precise protein structure determination of multi-domain proteins (Vitali et al 2006;.…”