Crystallization and preliminary X-ray analysis of Saccharomyces cerevisiae Ypd1p, a key intermediate in phosphorelay signal transduction

Abstract

Ypd1p, a 167-residue protein from Saccharomyces cerevisiae, plays a key role in osmosensing phosphorelay signal transduction. It forms part of a multistep phosphorelay system which also includes Sln1p hybrid histidine kinase and two response regulators, Ssk1p and Skn7p. It has been overexpressed in soluble form in Escherichia coli with a His 6 -tag at its C-terminus. The recombinant protein has been crystallized at room temperature using ammonium sulfate and lithium sulfate as precipitants. Native diffraction data have been collected to 2.3 A Ê using synchrotron radiation. The crystals are triclinic, belonging to the space group P1, with unit-cell parameters a = 65. 78, b = 66.74, c = 65.75 A Ê , = 106.60, = 106.48, = 115.53 . The asymmetric unit contains four molecules of the monomeric recombinant Ypd1p, with a corresponding V m of 2.75 A Ê 3 Da À1 and a solvent content of 55.3%.

Crystal structure of Escherichia coli CyaY protein reveals a previously unidentified fold for the evolutionarily conserved frataxin family

“…Overexpression, purification, and crystallization of the E. coli CyaY protein with a six-histidine tag at the C terminus were reported elsewhere (14). The dynamic light scattering analysis was performed with a Model DynaPro-801 instrument from Protein Solutions (Charlottesville, VA) as described (15).…”

Insights into eukaryotic multistep phosphorelay signal transduction revealed by the crystal structure of Ypd1p from Saccharomyces cerevisiae

“…This may serve an important function such as protein-protein interaction but its exact role remains to be established. 0Expression, puri®cation, crystallization, and X-ray data collection of Ypd1p fused with a His 6 tag at the C terminus were performed as described (Lee et al, 1999). Crystals belong to the triclinic space group P1 with the cell parameters of a 66.34 A Ê , b 66.48 A Ê , c 66.49 A Ê , a 106.4 , b 106.7 , and g 115.3 .…”