A Back Hydrogen Exchange
Procedure via the Acid-Unfolded
State for a Large Protein
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Abstract
A deuterated protein sample is required for nuclear magnetic
resonance
(NMR) measurements of a large protein because severe signal broadenings
occur because of the high molecular weight. The deuterated sample
expressed in <sup>2</sup>H<sub>2</sub>O should subsequently be subjected
to a back hydrogen exchange at amide groups. To perform the back exchange,
the protein molecule is unfolded or destabilized so that internal
residues become accessible to the solvent. However, the refolding
yield from the destabilized or unfolded state of a large protein is
usually low, leading to a dilemma in NMR measurements of large proteins.
In our previous paper [Suzuki, M., et al. (2011) <i>Biochemistry</i> <i>50</i>, 10390–10398], we suggested that an acid-denatured
microbial transglutaminase (MTG) consisting of 331 amino acid residues
can be recovered effectively under low-salt conditions, escaping from
the aggregation-prone intermediate. Here, we demonstrate that proMTG,
the pro form of MTG consisting of 376 amino acid residues, can be
refolded perfectly from the acid-unfolded state under low-salt conditions,
as confirmed by circular dichroism and NMR spectroscopies. By performing
the same procedure with a deuterated proMTG expressed in <sup>2</sup>H<sub>2</sub>O, we observed complete back exchanges for internal
residues by NMR spectroscopy. Our procedure has potential applications
to the back hydrogen exchange of large proteins for NMR measurements