| Autor: |
Loscha KV; Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia., Otting G |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of biomolecular NMR [J Biomol NMR] 2013 Jan; Vol. 55 (1), pp. 97-104. Date of Electronic Publication: 2012 Nov 29. |
| DOI: |
10.1007/s10858-012-9690-x |
| Abstrakt: |
Stereospecific resonance assignments of the α-protons of glycine are often difficult to obtain by measurements of scalar coupling constants or nuclear Overhauser effects. Here we show that these stereospecific resonance assignments can readily be obtained by cell-free protein synthesis in D(2)O, as the serine hydroxymethyltransferase, that is naturally present in E. coli cell extracts, selectively replaces the pro-2S proton of glycine by a deuterium. To encourage the conversion by serine hydroxymethyltransferase, we performed the cell-free reaction without the addition of any glycine, exploiting the capability of the enzyme to convert serine to glycine with the help of tetrahydrofolate. (13)C-HSQC spectra of ubiquitin produced with (13)C/(15)N-serine showed that about a quarter of the glycine residues derived from serine were stereospecifically deuterated. Pulse sequences are presented that select the signals from the stereospecifically deuterated glycine residues. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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