Investigations on gravity influence upon protein crystallization by the gel acupuncture technique

Most investigations on biological macromolecules are important for the knowledge of the functions in living organisms. Nowadays it is well known that the three-dimensional structure of proteins is obtained either by NMR or X-ray crystallographic methods. The crucial part in the latter is the availability of high-quality crystals in order to perform structural characterization. Nevertheless, there are some approaches to overcome this problem from the statistical [Jancarik and Kim, J. Appl. Crystallogr. 24 (1991) 409] and physico-chemical point of view [Riès-Kautt and Ducruix, Methods in Enzymology 276 Part A, Ch. 3, 1997, p. 23]. Once the crystals are obtained, the following part of the research must be focused on growing the crystal in order to have an appropriate size for X-ray analysis. There are additional advances in the methods for crystallizing, growing and determining what kind of biophysical or biochemical parameters have to be taken into account in order to obtain a high quality protein crystal, these advances have been already published elsewhere [Ducruix and Giegé, Crystallization of Nucleic Acids and Proteins. A Practical Approach, IRL Press, Oxford, 1991; McPherson, The Preparation and Analysis of Protein Crystals, Wiley, New York, 1982]. In order to evaluate these parameters, we have developed a new technique, called the gel acupuncture technique for crystallizing proteins inside an X-ray capillary tube as well as for trying to study the "in situ" crystal growth phenomena [García-Ruiz et al. Mater. Res. Bull. 28 (1993) 541; García-Ruiz and Moreno, Acta Crystallogr. D 50 (1994) 484]. In this work, we present our recent investigations on the influence of the gravity vector upon protein crystallization. Three proteins were chosen in order to test this possible influence, taking into account the size of each: satellite tobacco mosaic virus (1000 kDa) and two proteins of "low molecular weight", thaumatin I (22 kDa) and concanavalin A (200 kda). The experimental set-up was the same for the gel acupuncture technique [García-Ruiz et al. Mater. Res. Bull. 28 (1993) 541] and the experiments were carried out ranging from 0° (parallel) to 180° (anti-parallel) to the gravity vector to check its influence on protein crystallization. Thus, the counter-diffusion phenomena in protein crystallization were evaluated from parallel, 45°, 90° up to anti-parallel transport of the protein molecules to the gravity vector. Finally, we discussed why the molecular weight of the protein system should be taken into account in order to avoid a sedimentation phenomena and how the crystal growth technique is determined in order to keep the same diffusion pattern throughout the experiment.