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 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.
Wu
Peter S C
PS
Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia.
Ozawa
Kiyoshi
K
Jergic
Slobodan
S
Su
Xun-Cheng
XC
Dixon
Nicholas E
NE
Otting
Gottfried
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D002474:Cell-Free System; D004258:DNA Polymerase III; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D014157:Transcription Factors; C041273:transcription factor tau; D004258:DNA Polymerase III