Cytochrome c is one of the most studied proteins probably due to its electron-transfer properties in aerobic and anaerobic respiration. Particularly, cytochrome c from bovine heart is a small protein, M(r) 12,230 Da, globular (hydrodynamic diameter of 3.4 nm), soluble in different buffer solutions, and commercially available. Despite being a quite well-studied protein and relatively easy to manipulate from the biochemical and electrochemical viewpoint, its 3D structure has never been published. In this work, the purification, crystallization and 3D structure of one of the cytochrome c isoforms is presented to 1.5 A resolution. It is also shown how the presence of isoforms made both the purification and crystallization steps difficult. Finally, a new approach for protein electrocrystallization and design of biosensors is presented.

Ovocleidin-17 (OC17) from Gallus gallus is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. Here, the crystal structure of monomeric OC17, determined at a resolution of 1.5 Å, was refined to a crystallographic R-factor of 20.1%. This is the first protein directly involved in a non-pathological biomineralization process resolved by x-ray diffraction to date. The protein has a mixed ␣/␤ structure containing a single C-type lectinlike domain. However, although OC17 shares the conserved scaffold of the C-type lectins, it does not bind carbohydrates. Nevertheless, in vitro OC17 modifies the crystalline habit of calcium carbonate (CaCO 3 ) and the pattern of crystal growth at intervals of 5-200 g/ml. Determining the three-dimensional structure of OC17 contributes to a better understanding of the biological behavior of structurally related biomolecules and of the mechanisms involved in eggshell and other mineralization processes.

In this work we explore the possibilities of the gelacupuncture technique, proposed previously for the growth of protein single crystals [Garcia-Ruiz, Moreno, Viedma & Coll (1993). Mater. Res. Bull. 28,[541][542][543][544][545][546]. The main advantage of the technique is that the crystals are obtained inside an X-ray capillary and, unlike classical microdiffusion techniques, it involves a very simple and accurate technical arrangement that permits the continuous monitoring of the crystals in their growth environment. In particular, we describe the growth of single crystals of lysozyme, concanavalin A and ribonuclease A. Different starting conditions have been used to grow single crystals of these proteins into different types of capillaries at several protein and precipitating-agent concentrations. It is demonstrated that the technique works for a wide range of precipitating agents commonly used in protein crystal growth, such as large polymers (PEG 4000 and PEG 6000), organic solvents (from methanol to butanol) and salts [NaC1, (NH4)2SO4]. The range of inner diameter of the capillaries for which the technique works correctly has been also studied. The growth process and possible crystal movement was followed by video microscopy. Lysozyme crystals up to 3.1 mm were obtained but the average maximum linear crystal sizes were 2.0 mm for lysozyme, 0.4 mm for concanavalin A and 1.2 mm for ribonuclease, respectively. The waiting times to reach such a size, measured from the set-up of the experiments, were 72 h, 10 d and 5 d, respectively. Gels of tetramethoxysilane, tetraethoxysilane, sodium silicate, agar, high-strength agar and gel-gro have been tested in relation to their mechanical properties and their chemical interaction with the reactants. Finally, we discuss briefly the advantages of the gel-acupuncture technique and plausible applications other than crystal growth.

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