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J. Biochem, 2004, Vol. 135, No. 1 17-24
© 2004 The Japanese Biochemical Society

BIOCHEMISTRY

Mass Spectrometry on Segment-Specific Hydrogen Exchange of Dihydrofolate Reductase

Tatsuya Yamamoto, Shunsuke Izumi and Kunihiko Gekko*

Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526

To address the effects of local structures on structural fluctuations of Escherichia coli dihydrofolate reductase (DHFR), the backbone-fluctuation map was determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) coupled with H/D exchange and pepsin digestion. H/D exchange kinetics was examined at 15°C with 18 identified digestion fragments covering almost the entire amino acid sequence of DHFR. These fragments exhibited significant variations in the first-order rate constant of proton exchange, kex (0.47–0.71 min–1), the fraction of deuterium incorporation at the initial stage, Do (0.20–0.60), the fraction of deuterium incorporation at infinite time, D{infty} (0.75–0.97), and the number of protons protected from exchange, P (0.4–4.7), relative to the corresponding values for the whole DHFR molecule (kex = 0.51 min–1, Do = 0.41, D{infty} = 0.85, and P = 20.7). H/D exchange was very fast in the fragment comprising residues 5–28 (Met20 loop), which participates in substrate uptake, and reasonably fast in disordered and hydrophobic fragments, but slow in ß-strand-rich fragments. These results indicate that the local structures contribute differently to the fluctuation of the DHFR molecule, and that mass spectrometry coupled with H/D exchange and protease digestion is a useful tool for detecting segment-dependent protein fluctuation.

* To whom correspondence should be addressed. Fax: +81-824-24-7387; E-mail: gekko{at}sci.hiroshima-u.ac.jp


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