Microseeding - a powerful tool for crystallizing proteins complexed with hydrolyzable substrates.
Hydrolysis is an often-encountered obstacle in the crystallization of proteins complexed with their substrates. As the duration of the crystallization process, from nucleation to the growth of the crystal to its final size, commonly requires several weeks, non-enzymatic hydrolysis of an "unstable" ligand occurs frequently. In cases where the crystallization conditions exhibit non neutral pH values this hydrolysis phenomenon may be even more pronounced. ChoX, the substrate binding protein of a choline ABC-importer, produced crystals with its substrate acetylcholine after one month. However, these crystals exhibited only choline, an acetylcholine hydrolysis product, in the binding site. To overcome this obstacle we devised a microseeding protocol leading to crystals of ChoX with bound acetylcholine within 24 hours. One drawback we encountered was the high twinning fraction of the crystals, possibly was due to the rapid crystal growth.
Oswald
Christine
C
Institute of Biochemistry, Heinrich Heine University Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany.
Smits
Sander H J
SHJ
Institute of Biochemistry, Heinrich Heine University Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany.
Bremer
Erhard
E
Laboratory for Microbiology, Department of Biology, Philipps University Marbrug, Karl-von-Frisch Str., 35032 Marburg, Germany.
Schmitt
Lutz
L
Institute of Biochemistry, Heinrich Heine University Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany.