Mass Spectrometry on Segment-Specific Hydrogen Exchange of Dihydrofolate Reductase
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- Yamamoto Tatsuya
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University
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- Izumi Shunsuke
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University
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- Gekko Kunihiko
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University
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Description
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 (0.47-0.71min-1), the fraction of deuterium incorporation at the initial stage, Do (0.20-0.60), the fraction of deuterium incorporation at infinite time, D_??_ (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=0.51min-1, Do=0.41, D_??_=0.85, and P=20.7). H/D exchange was very fast in the fragment comprising residues 5-28 (Met 20 loop), which participates in substrate uptake, and reasonably fast in disordered and hydrophobic fragments, but slow in β-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 segmentdependent protein fluctuation.
Journal
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- The Journal of Biochemistry
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The Journal of Biochemistry 135 (1), 17-24, 2004
The Japanese Biochemical Society
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Details 詳細情報について
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- CRID
- 1390001204964022272
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- NII Article ID
- 130003418124
- 10016197305
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- NII Book ID
- AA00694073
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- COI
- 1:CAS:528:DC%2BD2cXjt1SmurY%3D
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- ISSN
- 17562651
- 0021924X
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- NDL BIB ID
- 6868337
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- PubMed
- 14999005
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- PubMed
- CiNii Articles
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- Abstract License Flag
- Disallowed