The taming of small heat-shock proteins: crystallization of the alpha-crystallin domain from human Hsp27

Abstract

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones. sHsps function as homooligomers or heterooligomers that are prone to subunit exchange and structural plasticity. Here, a procedure for obtaining diffraction-quality crystals of the alpha-crystallin domain of human Hsp27 is presented. Initially, limited proteolysis was used to delineate the corresponding stable fragment (residues 90-171). This fragment could be crystallized, but examination of the crystals using X-rays indicated partial disorder. The surface-entropy reduction approach was applied to ameliorate the crystal quality. Consequently, a double mutant E125A/E126A of the 90-171 fragment yielded well ordered crystals that diffracted to 2.0 A resolution.

Figure 1

Sequence alignment of human Hsp27, human Hsp22 and two T. saginata Tsp36 α-crystallin domains (ACD1 and ACD2). The alignment was produced using ClustalW (Thompson et al., 1994 ▶) with minor manual editing. The conserved regions are shaded in grey. Potential trypsin-cleavage sites after Arg and Lys residues in the Hsp22 and Hsp27 sequences are highlighted in yellow, negatively charged residues are marked in blue and Cys residues are shaded in mossy green. Purified proteolytic fragments of Hsp27 are boxed. The secondary-structure elements shown are as in the Tsp36 crystal structure. Residues Glu125 and Glu126 of the Hsp27 sequence that were chosen for mutagenesis are marked with asterisks.

Figure 2

Kinetics of Hsp27 trypsinolysis analyzed by SDS–PAGE. Lanes 1–3, Hsp27 after 30, 40 and 60 min digestion, respectively. Lane 4, original Hsp27 sample. Lane 5, molecular-weight markers (labelled in kDa).

Figure 3

Crystallization and diffraction patterns of the wild-type and mutated Hsp27 90–171 fragments. (a) Crystals of the wild-type fragment (maximum dimensions 0.17 × 0.08 × 0.05 mm). (b) Crystals of the E125A/E126A fragment (maximum dimensions 0.3 × 0.1 × 0.1 mm). (c) X-ray diffraction pattern from the WT fragment crystals showing partial disorder. (d) Diffraction pattern from the E125A/E126A crystals indicating a well ordered crystal lattice. (e) Indexing of the diffraction pattern shown in (c) assuming a hexagonal lattice with a = 76.0, c = 63.1 Å.