title: 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 H(2)O, exchange reactions can lead to contamination of (2)H sites by (1)H 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 (1)H 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 [U-(2)H, (15)N]-chlorella ubiquitin without and with added inhibitors, and [U-(15)N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-(13)C, (15)N]-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 C(α) sites, with the exception of Gly, and at C(β) 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-H(β), Asp-H(β), Gln-H(γ), Glu-H(γ), and Lys-H(ε). 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.
authors: National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry, University of Wisconsin-Madison, USA.
authors: Marco
authors: M
authors: Tonelli
authors: Kiran K
authors: KK
authors: Singarapu
authors: Shin-ichi
authors: S
authors: Makino
authors: Sarata C
authors: SC
authors: Sahu
authors: Yuko
authors: Y
authors: Matsubara
authors: Yaeta
authors: Y
authors: Endo
authors: Masatsune
authors: M
authors: Kainosho
authors: John L
authors: JL
authors: Markley
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D002474:Cell-Free System; D002708:Chlorella; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin
chemical_list: D000596:Amino Acids; D011506:Proteins; D025801:Ubiquitin; D006859:Hydrogen; D003903:Deuterium