Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions

Abstract

GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 A crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes that may occur in response to cognate potassium-ion binding by comparison to the previously determined wild-type GroEL structure (PDB code 1xck), in which potassium ions were observed in all 14 subunits. In general, the structure is similar to the previously determined wild-type GroEL crystal structure with some differences in regard to temperature-factor distribution.

Figure 1

Photograph of a GroEL crystal illuminated with polarized light. The crystals in this photograph grew to final size within one week at 277 K in a crystallization solution consisting of 100 mM imidazole pH 8 containing 34%(v/v) MPD and 0.32 M MgCl₂. The scale bar represents ∼200 µm.

Figure 2

Image of a GroEL monomer showing the location of the potassium ion-binding site as well as the regions that were identified as being most flexible in a comparison to the previously determined wild-type GroEL structure (PDB code 1xck). Residues 44–45, 202–204 and 260–268 line the folding cavity, while residues 477–487 are located on the outside surface near the ATP-binding site and ring–ring interface of the GroEL complex. This figure was generated using PyMOL v.0.99 (DeLano, 2002 ▶).