caa a22 4500 445325364 CHVBK 20180317142718.0 cr unu---uuuuu 170323e20111201xx s 000 0 eng 10.1007/s10858-011-9575-4 doi (NATIONALLICENCE)springer-10.1007/s10858-011-9575-4 Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition [Elektronische Daten] [Marco Tonelli, Kiran Singarapu, Shin-ichi Makino, Sarata Sahu, Yuko Matsubara, Yaeta Endo, Masatsune Kainosho, John Markley] 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 (1mM aminooxyacetic acid) and glutamate synthetase (0.1mM l-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-2H, 15N]-chlorella ubiquitin without and with added inhibitors, and [U-15N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-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: Asn-Hβ, Asp-Hβ, Gln-Hγ, Glu-Hγ, and Lys-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. Springer Science+Business Media B.V., 2011 Cell-free translation nationallicence Chlorella ubiquitin nationallicence SAIL nationallicence Perdeuterated protein nationallicence Proton back exchange nationallicence Transamination nationallicence Transaminase inhibitor nationallicence Tonelli Marco National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut Singarapu Kiran National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut Makino Shin-ichi Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut Sahu Sarata Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut Matsubara Yuko Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut Endo Yaeta Cell-Free Science and Technology Research Center, Ehime University, 790-8577, Matsuyama, Japan aut Kainosho Masatsune Center for Priority Areas, Tokyo Metropolitan University, 192-0397, Minami-ohsawa, Hachioji, Tokyo, Japan aut Markley John National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut Journal of Biomolecular NMR Springer Netherlands 51/4(2011-12-01), 467-476 0925-2738 51:4<467 2011 51 10858 https://doi.org/10.1007/s10858-011-9575-4 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9575-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Tonelli Marco National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut NATIONALLICENCE 700 1- Singarapu Kiran National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut NATIONALLICENCE 700 1- Makino Shin-ichi Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut NATIONALLICENCE 700 1- Sahu Sarata Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut NATIONALLICENCE 700 1- Matsubara Yuko Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut NATIONALLICENCE 700 1- Endo Yaeta Cell-Free Science and Technology Research Center, Ehime University, 790-8577, Matsuyama, Japan aut NATIONALLICENCE 700 1- Kainosho Masatsune Center for Priority Areas, Tokyo Metropolitan University, 192-0397, Minami-ohsawa, Hachioji, Tokyo, Japan aut NATIONALLICENCE 700 1- Markley John National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, 53706-1549, Madison, WI, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 51/4(2011-12-01), 467-476 0925-2738 51:4<467 2011 51 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer