A back hydrogen exchange procedure via the acid-unfolded state for a large protein.
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 (2)H(2)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) Biochemistry 50, 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 (2)H(2)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.
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Osaka 565-0871, Japan. mototaka_suzuki@ajinomoto.com
Mototaka
M
Suzuki
Kazumasa
K
Sakurai
Young-Ho
YH
Lee
Takahisa
T
Ikegami
Keiichi
K
Yokoyama
Yuji
Y
Goto
D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003903:Deuterium; D004792:Enzyme Precursors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D058849:Protein Refolding; D058767:Protein Unfolding; D013302:Streptomyces / Q000201:enzymology*; D011503:Transglutaminases / Q000737:chemistry*
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D003903:Deuterium; D011503:Transglutaminases