**6. References**


Due to the inherent difficult of make crystals of proteins, particularly for large proteins like the *G. paulistus*, the presented results demonstrated the capability of SAXS technique and the new optimization methods to provide a fast and reliable procedure to investigate the shape and quaternary structures of large protein complexes. Also, the correlation of SAXS results with the hydrodynamic properties increases the reliability of the results and makes

General aspects of Small Angle X-Ray applied to the study of colloidal particles in solution were presented. Two examples of application were shown, demonstrating the versatility of the SAXS technique. One of the main strengths of this technique is the possibility of investigate systems directly in solution, close to the native state, in a broad range of sizes and molecular weights. On the other hand, due to the low information content of a typical SAXS data, the scattering data has to be correlated and supported by additional information, obtained from other experimental techniques. In this way, even thought SAXS data can provide a valuable and important structural information, the technique and the modeling methods has to be applied with extreme precaution and always cross checked with several additional results in order to provide relevant, unambiguous information and, most importantly, avoid wrong data interpretation. As shown in this chapter, absolute scale calibration and the comparison of hydrodynamic properties of the obtained models with the ones obtained experimentally are two very useful tools for results checking and model

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**4. Conclusions** 

validation.

**5. Acknowledgment** 

several valuable discussions.

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**17** 

*Italy* 

**Monitoring Preparation of Derivative Protein** 

*Department of Chemistry, University of Naples "Federico II", Naples, Italy Istituto di* 

Below to crystallographic applications, protein crystals are of great interest in other numerous fields of biology and biotechnology. Cataract, or the loss of transparency of the eye lens, is related to the alteration of physical, chemical, and structural properties of proteins of the crystallin family, that may lead to crystallization under some physiological conditions (Tardieu, 1998). Other pathological states are known to be a consequence of the *in vivo* formation of crystals, made of either proteins or other macromolecular assemblies. Examples are viral proteins stored in plant cells, viral particles in animal cells, hemoglobin C and S causing anemia, or ribosomal particles accumulating in the brain of patients suffering

Of course, crystals of biological macromolecules that are prepared *in vitro* have important applications: they are tools to obtain atomic models of the molecules and to design specific ligands and new drug formulations. Medicinal formulations composed of either insulin (Richards et al., 1999) or α-interferon crystals (Reichert et al., 1996) are already applied in treatments to ensure the continuous release of protein in blood. Crystallographic analysis of highly ordered crystals with intense X-ray sources provides accurate three dimensional structures (Ducruix & Giegé, 1999). The success of this technique strictly depends on obtaining diffraction-quality crystals. The process of crystallization remains a hit-and-miss affair, typically involving screening hundreds of conditions. The crystallization of biological macromolecules shares many common properties with those of small solute molecules (e.g. growth by 2D nucleation or by screw dislocation mechanisms), but their crystals exhibit several peculiarities: most of them have a high solvent content (e.g. 30-80 vol%), few

Briefly, protein crystallization requires the formation of a supersaturated protein-precipitant solution. The transition from a stable solution to a supersaturated one can be achieved by increasing the concentration of precipitant and/or that of protein (Vergara et al., 2003). The most frequently used crystallization method is the *vapor diffusion technique*. A drop containing protein, buffer, salt and precipitant is equilibrated against a reservoir (buffer, salt and precipitant). The difference in concentration between the drop (lower) and the reservoir (higher) drives the system toward equilibrium by diffusion through the vapor phase. The drop can either be placed on the underside of the cover slide (*hanging drop*) or placed on a

intermolecular contacts, and a high density of defects (Malkin et al., 1996).

**1. Introduction** 

from *presenile dementia* (McPherson, 1999).

**Crystals** *via* **Raman Microscopy** 

*Biostrutture e Bioimmagini, CNR, Naples* 

Antonello Merlino, Filomena Sica and Alessandro Vergara\*

