Investigations on protein crystal growth by the gel acupuncture method

Abstract

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.

Effect of an External Electric Field on the Kinetics of Dislocation-Free Growth of Tetragonal Hen Egg White Lysozyme Crystals

“…The growth of high-quality single crystals of proteins has been intensively pursued using magnetic fields [3][4][5][6][7][8][9], microgravity [10][11][12][13][14][15][16], solution flow [17][18][19][20], and gel as a growth host media [21][22][23][24][25][26][27][28]. A growth technique mediated by screw dislocations under low supersaturation has also been proposed [29][30][31][32].…”

Trends and Challenges in Experimental Macromolecular Crystallography

“…The combination of that approach with growth of larger crystals via novel crystallization geometries (see e.g. Garcia-Ruiz & Moreno, 1994) and/or microgravity can considerably extend the scope of neutrons to many more protein systems, and so overcome the principal weakness of X-rays, namely, the problem of location of hydrogens, and which is a large area of enzymology.…”

Growth of shaped single crystals of proteins

“…The experimental set-up utilized for the growth of protein single crystals was typical of the gel acupuncture method [6,7]. The layers of silica gel were prepared in crystallization vessels of 25 mm in diameter and 60 mm high.…”