Mass spectrometry on segment-specific hydrogen exchange of dihydrofolate reductase.
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 degrees 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, k(ex) (0.47-0.71 min(-1)), the fraction of deuterium incorporation at the initial stage, D(o) (0.20-0.60), the fraction of deuterium incorporation at infinite time, D(infinity) (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 (k(ex) = 0.51 min(-1), D(o) = 0.41, D(infinity) = 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 beta-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.
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526.
Tatsuya
T
Yamamoto
Shunsuke
S
Izumi
Kunihiko
K
Gekko
D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
D010446:Peptide Fragments; D013762:Tetrahydrofolate Dehydrogenase