Expression, crystallization and crystallographic analysis of DegS, a stress sensor of the bacterial periplasm.
Regulated proteolysis is a key event in transmembrane signalling between intercellular compartments. In Escherichia coli, a protein DegS has been identified as being a periplasmic stress sensor for unfolded or misfolded outer membrane proteins (OMPs). Activation of DegS initiates a proteolytic cascade which results in the transcription of periplasmic genes under sigmaE control, most importantly chaperones and proteases. DegS has been cloned and expressed as full-length protein and in an N-terminally truncated form. Both proteins were tested for crystallization and two forms of well diffracting crystals of the truncated form were obtained. Crystals of form I diffract to 3.5 A and belong to space group P2(1)3, while crystals of form II diffract to 2.2 A and belong to space group I23. Crystals of form II were soaked with a consensus peptide representing the C-termini of outer membrane proteins and data to 2.4 A resolution were collected. Molecular-replacement trials using a homologous protease domain indicate the presence of two molecules in the asymmetric unit of crystal form I. The correctness of the molecular-replacement solution was verified by identifying radiation-damage-induced structural changes.
Grininger
Martin
M
Max-Planck-Institute for Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Ravelli
Raimond B G
RB
Heider
Ursel
U
Zeth
Kornelius
K
D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D019897:Periplasm / Q000737:chemistry*
D029968:Escherichia coli Proteins; C489349:degS protein, Escherichia coli