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Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus
Agapay, R.C.; Savvides, S.N.; Van Driessche, G.; Devreese, B.; Van Beeumen, J.; Jongejan, J.A.; Hagen, W.R. (2005). Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus. Acta Crystallographica Section F-Structural Biology Communications 61: 914-916. dx.doi.org/10.1107/S1744309105028393
In: Acta Crystallographica Section F-Structural Biology Communications: Chester. ISSN 2053-230X, more

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Keyword
    Marine

Authors  Top 
  • Agapay, R.C.
  • Savvides, S.N.
  • Van Driessche, G.
  • Devreese, B.
  • Van Beeumen, J., more
  • Jongejan, J.A.
  • Hagen, W.R.

Abstract
    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 Å 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 Å, 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.

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