Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus.
The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8 A using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8 A, respectively, using synchrotron radiation. Both the untreated and EPNP-treated crystals crystallize isomorphously in space group C2 and contain three dimers in the asymmetric unit. The PMSF-treated crystals also belong to this space group and have almost identical packing density, but show dramatically different unit-cell parameters.
Agapay
Ramelito C
RC
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Savvides
Savvas N
SN
Van Driessche
Gonzalez
G
Devreese
Bart
B
Van Beeumen
Jozef
J
Jongejan
Jaap A
JA
Hagen
Wilfred R
WR
D016880:Anisotropy; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004852:Epoxy Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D006868:Hydrolysis; D008024:Ligands; D009596:Nitrophenols / Q000737:chemistry; D010664:Phenylmethylsulfonyl Fluoride / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry; D014158:Transcription, Genetic; D014961:X-Ray Diffraction
C509334:AsnC protein, E coli; D004852:Epoxy Compounds; D029968:Escherichia coli Proteins; D008024:Ligands; D009596:Nitrophenols; D015534:Trans-Activators; D010664:Phenylmethylsulfonyl Fluoride; C007606:1,2-epoxy-3-(p-nitrophenoxy)propane