Porous silicon: a nucleation-inducing material for protein crystallization

Abstract

The anaphase-promoting complex (APC) is a large multi-subunit complex that has cell cycle regulated ubiquitin-protein ligase (E3) activity. Together with an ubiquitin activating (E1) and an ubiquitin-conjugating enzyme (E2) the APC catalyses the formation of polyubiquitin chains on substrate proteins such as securin and mitotic cyclins and thereby targets them for the degradation by the 26S proteasome. The human APC is composed of at least 11 subunits. For understanding why the APC is assembled of so many subunits it will be of importance to know the structure of the complex. Structural information is available only for APC10/DOC1 whose crystal structure has been solved recently. However, no crystal structure is shown for the other 10 APC subunits and crystallization of holo APC is currently impossible due to the lack of appropriate purification protocols. To gain insight to the structure of the holo APC, we purified HeLa cell APC in a homogeneous soluble form suitable for electron microscopic analyses. Based on the analysis of 13000 molecular images we obtained a three-dimensional model of the human APC at a resolution of 24 Å. The APC has a complex asymmetric structure of about 150 Å in diameter in which an outer protein wall encloses an inner cavity that could represent a reaction chamber for ubiquitination reactions. For further analyses we developed a method that allows the purification of a large APC subcomplex. Structural and biochemical analyses of this sub-complex should help to understand the function and localization of individual subunits within the holo APC. Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response including G2/M cell cycle control. Staurosporine-like kinase inhibitors, UCN-01 (7-hydroxystaurosporine) and SB218078 have been shown to be potent Chk1 inhibitors that abrogate the G2/M checkpoint induced by DNA damaging agents. To understand the structural basis for Chk1 regulation and inhibition by UCN-01 and its analogs, we determined the crystal structures of Chk1-staurosporine, Chk1-UCN-01 and Chk1-SB218078 binary complexes. Keywords: UBIQUITINATION CRYOELECTRONMICROSCOPY CELLCYCLEThe structure shows that all three compounds bind to the ATP binding pocket of Chk1, and cause only slight changes in the protein conformation. The high structural complementarity of these interactions is consistent with the potency and selectivity of this class of inhibitors. X-ray crystallography has also revealed some specific binding interactions of inhibitors to Chk1. We made a comparison between these Chk1 complexes and cyclin-dependent kinases 2 (CDK2) complexed with staurosporine, in respect of both structure and biological activity of the inhibitors to Chk1 and CDKs. Selectivity of UCN-01 towards Chk1 over CDKs can be explained by the presence of hydroxyl group in the lactam moiety interacting with the ATP binding pocket. Production of high quality protein crystals of both soluble and especially of membrane proteins remains a difficult and important task. Hence there...

Protein crystallization facilitated by molecularly imprinted polymers

“…13); however, this requires the availability of crystals of the given protein or at least some crystalline material to start with. In an ongoing search for alternative heterogeneous seeding materials, a variety of substances such as minerals (14), horse (15) and human (16) hair, thin films (17), charged surfaces (18,19), mesoporous materials (20)(21)(22), and other materials (23) have been used as nucleants with varied success. The problem with such materials is that they are random substances, which have helpful properties such as porosity, nanostructure, or electrostatic attractive potential, but no designed specificity for proteins.…”

Is Ice Nucleation from Supercooled Water Insensitive to Surface Roughness?

“…[11][12][13][14] Highly porous materials can also be extremely efficient nucleators, but this requires pores of optimal diameters. [15][16][17][18][19][20] Studies of crystal nucleation from vapour have provided even more conclusive evidence for the involvement of topography.…”

Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

“…Recent studies demonstrate that porous materials, such as porous silicon, with porous sizes comparable to the size of the protein molecules, display nucleation-inducing properties. Specific experimental methods for cross-seeding and epitaxial are not different from the ones already detailed, therefore apply the approach described in Steps 74-97 [48][49][50][51]106 .…”

“…Different mineral samples (i.e., mica and apophyllite) may be used as heteroepitaxial nucleant. Recent studies demonstrated that porous materials (i.e., porous silicon), containing pore sizes that are comparable with the size of the protein molecules, display nucleation-inducing properties [48][49][50][51] .…”