Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Oct 1;61(Pt 10):914-6. doi: 10.1107/S1744309105028393. Epub 2005 Sep 30.

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 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.

MeSH terms

  • Anisotropy
  • Crystallization
  • Crystallography, X-Ray
  • Dimerization
  • Epoxy Compounds / chemistry
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Hydrolysis
  • Ligands
  • Nitrophenols / chemistry
  • Phenylmethylsulfonyl Fluoride / chemistry
  • Protein Structure, Tertiary
  • Pyrococcus furiosus / metabolism*
  • Trans-Activators / chemistry
  • Transcription, Genetic
  • X-Ray Diffraction

Substances

  • AsnC protein, E coli
  • Epoxy Compounds
  • Escherichia coli Proteins
  • Ligands
  • Nitrophenols
  • Trans-Activators
  • Phenylmethylsulfonyl Fluoride
  • 1,2-epoxy-3-(p-nitrophenoxy)propane