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Abstract: Protein crystallization is a powerful purification tool. It is the first step for crystallographic structural investigations, and can be preparatory for biotechnological applications. However, crystallizing proteins is challenging and methods to control the crystallization process are needed. Ionic-liquid hydrogel composite membranes (IL-HCMs) have been used here as material capable of supporting protein crystallization and hosting grown crystals. We found that IL-HCMs affect the selection mechanism of glucose isomerase (GI) polymorphs and make GI crystals grow completely immersed into the hydrogel layer. X-ray diffraction studies show that IL ions do not bind to the protein, likely because IL molecules are constrained in the polymeric framework. Our GI crystal structures have been compared with many existing GI crystal structures using multivariate analysis tools, allowing a comprehensive overview of factors determining structural similarities, i.e., temperature variations and external stresses exerted during or after crystal growth, such as dehydration or presence of hydrogel of a different nature. GI crystals grown on IL-HCM fit perfectly in this framework, showing typical features induced by external forces. Overall, protein crystallization by IL-HCMs show potential for biotechnological applications, as it could constitute a natural means for containing crystallized enzymes in working conditions.

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Abstract: In this study, we exploited the possibility of tuning physical–chemical properties of hydrogel composite membranes (HCMs) surfaces, by using iron oxide nanoparticles (NPs) as topographical designers, with the aim of examining the effect of surface topography and wettability on the heterogeneous nucleation of protein crystals. On the basis of roughness and contact angle measurements, it was found that surface structural characteristics, in addition to chemical interactions between the surface and protein molecules, have influence on the heterogeneous nucleation of lysozyme and thermolysin crystals to different extents. We demonstrated that increasing the amount of NPs incorporated in the hydrogel matrix promotes protein nucleation to a higher extent, potentially due to the increase of local solute concentration, arising from the enhanced wetting tendency in the Wenzel regime, and physical confinement at rougher hydrophilic surfaces. An extensive crystallographic analysis suggested the tendency of the growing crystals to incorporate hydrogel materials, which allows inducement of protein conformational states slightly different from those covered by standard crystallization methods. Protein flexibility can be thus sampled by changing the amount of NPs in the HCMs, with negligible influence on the quantity and quality of X-ray diffraction data.

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Abstract: Copper tra ffi cking proteins have been impli- cated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains mono- dentate ammines, oxaliplatin contains chelated 1,2-diaminocy- clohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH) 2+ } moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox1 2 · Pt 2+ 2 , indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have a ff orded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu + , Cd 2+ , and Hg 2+ ), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu + ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the reinterpretation of the original crystallographic data put forward by Shabalin et al. (1 Cu occupancy), and could apply to other cases.