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, pp. 94-98
Section 4.2.3. General properties of detergents relevant to membrane-protein crystallization
aMax-Planck-Institut für Biophysik, Heinrich-Hoffmann-Strasse 7, D-60528 Frankfurt/Main, Germany |
The presence of detergents sometimes causes problems. The monomeric detergent itself can crystallize, e.g. dodecyl-β-D-maltoside at 4 °C in the presence of polyethylene glycol. The detergent crystals might be mistaken for protein crystals. Detergent micelles possess attractive interactions (see Zulauf, 1991). Upon addition of salts or polyethylene glycol, or upon temperature changes, a phase separation may be observed: owing to an increase in these attractive interactions, the detergent micelles `precipitate', forming a viscous detergent-rich phase and a detergent-depleted aqueous phase. The membrane proteins are found exclusively in the viscous phase and crystals – if formed – are difficult to handle. Some detergents, e.g. those with polyoxyethylene head groups, undergo a phase separation at higher temperatures. This phenomenon has been used to separate solubilized membrane proteins, which are found in the detergent-rich phase, from the water-soluble proteins. The latter are concentrated in the detergent-depleted phase (Bordier, 1981
). Other detergents, e.g. octyl-β-D-glucopyranoside, show this phase separation at lower temperatures. Therefore, if phase separation causes problems, a change of the crystallization temperature may help.
The polar head groups of the detergents influence their usage in many ways. One would like to have a small polar head group, because the head group `covers' the part of the protein's polar surface that is adjacent to the hydrophobic surface belt. The bigger the head group the more of the polar surface is covered and unavailable for the polar interactions needed to form the crystal lattice. Unfortunately, detergents with small polar head groups are rather denaturing. Detergents with charged head groups cannot be used, but detergents with zwitterionic head groups, e.g. sulfobetaines, can be tried with more stable proteins. The head group of a very successful detergent, N,N-dimethyldodecylamine-N-oxide, is of zwitterionic nature. I estimate that it can only be used with about 20% of all membrane proteins. Detergents with sugar residues as head groups have been used successfully. Octyl-β-D-glucopyranoside also tends to be denaturing. The lifetime of many membrane proteins can be prolonged by a factor of three by the use of nonyl-β-D-glucopyranoside instead of the shorter homologue. Such behaviour is observed within each series of homologous detergents; an increase in the alkyl chain by one methylene group leads to an increase in stability by a factor of three, an increase by two methylene groups leads to an increase in stability by a factor of about ten. Unfortunately, decyl-β-D-glucopyranoside is too insoluble to be used as detergent. For less stable membrane proteins, alkylmaltoside detergents or alkanoylsucrose detergents have to be tried. There is one special problem when using alkyl-β-D-glucopyranosides or alkyl-β-D-maltosides as detergents: the commercially available detergents are often `contaminated' with the α-anomers in varying, sometimes substantial, concentrations. The α-anomers are much less soluble, and appear to prevent crystallization. In the case of photosystem I from a thermophilic cyanobacterium, it has been reported that for a 2% α-anomer content in dodecyl-β-D-maltoside preparations no crystals can be obtained, with a 0.5–2% content the diffraction of the crystals is anisotropic with a reduction in resolution to 5–6 Å, whereas diffraction to better than 3.5 Å resolution in all directions is observed when the content of the α-anomer is below 0.1% (Fromme & Witt, 1998). The percentage of α-anomer can be determined using NMR spectroscopy or high-performance liquid chromatography. During ageing of detergent solutions, a conversion from the β-anomer to the α-anomer is expected. Therefore, ageing of detergent solutions has to be prevented.
The detergents that have been successfully used to crystallize membrane proteins can also be found in Table 4.2.1.1. The possibilities for developing new detergents for membrane-protein crystallization have not been exhausted. There is a need for new detergents, e.g. detergents with head groups with sizes between glucose and maltose are still missing!
It has been observed that crystals of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis could be obtained when N,N-dimethyldodecylamine-N-oxide is used as detergent, but not when N,N-dimethyldecylamine-N-oxide is used. Even crystals formed with the dodecyl homologue lost their order when soaked in a buffer containing the decyl homologue. These observations were found to have an obvious explanation when the location of the detergent molecules bound to the protein was determined using neutron crystallography (Roth et al., 1989); the detergent molecules surrounding neighbouring photosynthetic reaction centres in the crystal lattice are in contact. It is likely that attractive interactions between neighbouring protein-bound detergent micelles contribute to the stability of the crystal lattice. Particularly striking (see Table 4.2.3.1
) is the dependence of the crystal quality on the alkyl-chain length in the case of the two-subunit cytochrome c oxidase from the soil bacterium Paracoccus denitrificans. Well ordered crystals were obtained with undecyl-β-D-maltoside, but not with the decyl and dodecyl homologues. Table 4.2.3.1
also lists the names of important vendors of detergents.
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