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Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition


Abstract Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H2O, exchange reactions can lead to contamination of 2H sites by 1H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing 1H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM l-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [Uâ??2H, 15N]-chlorella ubiquitin without and with added inhibitors, and [Uâ??15N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [Uâ??13C, 15N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at Cα sites, with the exception of Gly, and at Cβ sites of Ala. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Asnâ??Hβ, Aspâ??Hβ, Glnâ??Hγ, Gluâ??Hγ, and Lysâ??Hε. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of interest in studies of large proteins, protein complexes, and membrane proteins.

• Content Type Journal Article
• Category Article
• Pages 1-10
• DOI 10.1007/s10858-011-9575-4
• Authors
  • Marco Tonelli, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA
  • Kiran K. Singarapu, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA
  • Shin-ichi Makino, Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA
  • Sarata C. Sahu, Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA
  • Yuko Matsubara, Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA
  • Yaeta Endo, Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, 790-8577 Japan
  • Masatsune Kainosho, Center for Priority Areas, Tokyo Metropolitan University, Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan
  • John L. Markley, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR