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International
Tables for Crystallography Volume F Crystallography of biological macromolecules Edited by M. G. Rossmann and E. Arnold © International Union of Crystallography 2006 |
International Tables for Crystallography (2006). Vol. F. ch. 4.2, p. 99
Section 4.2.7. General recommendations
aMax-Planck-Institut für Biophysik, Heinrich-Hoffmann-Strasse 7, D-60528 Frankfurt/Main, Germany |
When trying to crystallize a membrane protein the first, and most important, task is to obtain a pure, stable and homogeneous preparation of the membrane protein under investigation. Unfortunately, general methods for producing substantial amounts of membrane proteins using heterologous expression systems in a native membrane environment do not exist, nor is refolding of membrane proteins from inclusion bodies well established. Once a pure and homogeneous preparation of a membrane protein has been obtained, its stability in various detergents has to be investigated at different pH values. Frequently, a sharp optimum pH for stability is observed with detergents of shorter alkyl-chain length. In crystallization attempts, the usual parameters (nature of the precipitant, buffer, pH, addition of inhibitors and/or substrates, see Chapter 4.1![[link]](/graphics/greenarr.gif)
) should be varied. The most important variable, however, is the detergent. Detergents can be exchanged most conveniently by binding the membrane protein to column materials (e.g. ion-exchange resins, hydroxyapatite, affinity matrices), washing with a buffer containing the new detergent at concentrations above the CMC under non-eluting conditions, and then eluting the bound membrane protein with a buffer containing the new detergent. The completeness of the detergent exchange should be checked. For this purpose, the use of 14C- or 3H-labelled detergents is recommended. Washing with about 20 column volumes is frequently required for a complete detergent exchange. It is more difficult to exchange a detergent with a low CMC (this means long alkyl chains) against a detergent with a high CMC (shorter alkyl chains). The detergent can often be exchanged during a final step in the purification. Less satisfying methods for detergent exchange are molecular-sieve chromatography, ultracentrifugation or repeated ultrafiltration and dilution. If the amount of membrane protein is limited, it is advisable to restrict the usual parameters to a set of, e.g., 48 standard combinations with respect to precipitating agent, pH, buffer etc., but to try all available detergents. If crystals of insufficient quality or size are obtained, trying the antibody Fv fragment approach is highly recommended. Alternatives are to use a different source (species) for the membrane protein under investigation, or to remove flexible parts of the membrane protein by proteolytic digestion. I am convinced that 50% of all membrane proteins will yield well ordered, three-dimensional crystals within five years, once the problem of obtaining a pure, stable and homogeneous preparation has been solved.
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