Giovanna Tosi

Crystallization of proteins on functionalized surfaces

Functionalized mica sheets and polystyrene films exposing ionisable groups have been used as heterogeneous nucleating surfaces for model proteins. Surfaces with different densities of amino or sulfonated groups have been prepared. Crystallization trials were carried out using the hanging-drop vapour-diffusion method. The results show that using these surfaces the starting protein concentration necessary to form crystals is reduced. The effect of these surfaces on the crystallization process may be the consequence of electrostatic interactions between charged residues of proteins and ionisable groups on surfaces. These interactions can be attractive or repulsive, depending on the relative charge of the protein and the surface at the crystallization pH. Both phenomena can induce an increase of the local protein concentration on the surface or in its proximity, favouring nucleation. Moreover, a reduction of the waiting time (an estimation of the nucleation time) was also observed for some proteins, suggesting a surface-stabilization effect on crystal nuclei.

Structure/function studies on two type 1 ribosome inactivating proteins: Bouganin and lychnin

The three-dimensional structures of two type 1 RIPs, bouganin and lychnin, has been solved. Their adenine polynucleotide glycosylase activity was also determined together with other known RIPs: dianthin 30, PAP-R, momordin I, ricin A chain and saporin-S6. Saporin-S6 releases the highest number of adenine molecules from rat ribosomes, and poly(A), while its efficiency is similar to dianthin 30, bouganin and PAP-R on herring sperm DNA. Measures of the protein synthesis inhibitory activity confirmed that saporin-S6 is the most active. The overall structure of bouganin and lychnin is similar to the other considered RIPs and the typical RIP fold is conserved. The superimpositioning of their C(alpha) atoms highlights some differences in the N-terminal and C-terminal domains. A detailed structural analysis indicates that the efficiency of saporin-S6 on various polynucleotides can be ascribed to a negative electrostatic surface potential at the active site and several exposed positively charged residues in the region around that site. These two conditions, not present at the same time in other examined RIPs, could guarantee an efficient interaction with the substrate and an efficient catalysis.

Crystallization and preliminary X-ray diffraction data analysis of stenodactylin, a highly toxic type 2 ribosome-inactivating protein from Adenia stenodactyla

Ribosome-inactivating proteins (RIPs) inhibit protein synthesis and induce cell death by removing a single adenine from a specific rRNA loop. They can be divided into two main groups: type 1 and type 2 RIPs. Type 1 RIPs are single-chain enzymes with N-glycosidase activity. Type 2 RIPs contain two chains (A and B) linked by a disulfide bond. The A chain has RIP enzymatic activity, whereas the B chain shows lectin activity and is able to bind to glycosylated receptors on the cell surface. Stenodactylin is a type 2 RIP from the caudex of Adenia stenodactyla from the Passifloraceae family that has been recently purified and characterized. It shows a strong enzymatic activity towards several substrates and is more cytotoxic than other toxins of the same type. Here, the crystallization and preliminary X-ray diffraction data analysis of stenodactylin are reported. This RIP forms crystals that diffract to high resolution (up to 2.15 A). The best data set was obtained by merging data collected from two crystals. Stenodactylin crystals belonged to the centred monoclinic space group C2 and contained two molecules in the asymmetric unit.

Protein crystallization on nucleating surfaces inspired by biomineralization

The production of diffraction quality protein crystals remains one of the greatest impediments facing structural biologist today: it has always been a bottleneck to structure determination. The Bologna group had designed protein crystal nucleating substrates inspired by the studies on the biomineralization processes [1]. In biomineralization is well known the crystal surfaces are able to recognize and organize specific macromolecules, thus having potential nucleating properties. In the proposed protein crystal growth strategy the control processes over the crystal nucleation and growth occur by means of surfaces functionalized with specific ionisable groups and controlled hydrophilicity. Experimentally the traditional hanging-drop vapour diffusion technique was modified introducing between the glass cover slip and the drop, a surface opportunely functionalised with ionizable chemical groups. The used nucleant surfaces were silanized mica and sulphonated polystyrene films. Studies of contact angles, roughness, morphology (AFM, SEM) and ATR-FTIR demonstrated that both surfaces are substrates suitable for macromolecular crystallization: they are enough smooth to avoid topographic effects during nucleation, and their hydrophilicity still allows the deposition of a spherical drop. To evaluate the nucleant capacity of surfaces and understand the mechanism by which the surfaces control the protein crystallization, several experiments were carried out using three different “model proteins”: Thaumatin, Concanavalin A and Lysozyme [2]. The results showed that functionalised surfaces, when compared with siliconized glass cover slip (control), decreased the induction time and the starting protein concentration for the crystal nucleation and growth. An increase of the nucleation density with the increase of the ionisable group density on the surface was also observed. It is proposed that non-specific attractive and local interactions between the charged residues of the protein and the ionisable groups on surface might promote molecular collisions raising the probability of crystalline nucleus formation.