The Classical Approach

Integral membrane proteins are embedded in a lipidic membrane. In order to be crystallized in three dimensions, they have to be solubilized and purified from their native environment. Amphiphilic molecules such as detergent form a belt around the membrane proteins, in which the tails of the detergent protect the hydrophobic protein surface whereas the headgroups protrude toward the solvent. The crystallization was described in several reviews or book chapters (Hunte and Michel 2003; Reiss-Husson and Picot 1999). Neutron diffraction experiments revealed that three-dimensional crystals are built up of such mixed micelles and that crystal contacts leading to high-resolution diffracting crystals implicate protein-protein interactions (Roth et al. 1989). In a very crude approximation, it could be considered that the mixed micelle solution is similar to a solution of a soluble protein. Crystallization can therefore be obtained by adding the adequate précipitants and adjusting their concentrations in order to cross the solubility curve and to reach the metastable zone where crystallization occurs. In reality, the presence of detergent makes the process more complex as detergents have their own phase behaviour (Zulauf 1991). At low concentration they are solubilized in water as monomers. Above the critical micellar concentration (cmc) they aggregate in micelles in which hydrophilic (polar or charged) moieties are exposed to the solvent; these micelles coexist in solution with monomers. The phase diagram as a function of detergent concentration and temperature shows that at higher detergent concentrations two phases coexist; one is poor, the other is rich in detergent (Fig. 3.1).

The phase diagrams are influenced by the precipitant, its concentration and all other chemical components. Approaching the consolution boundary, from the micellar phase to the phase separation, enhances the micelle-micelle interactions and the probability of inducing protein-protein interactions. In addition, crossing the consolution boundary will concentrate the protein in the phase which is

Micelle Diagrams

Fig. 3.1. Schematic phase diagram for non-ionic detergent water system. The abscissa and ordinate are without scale. Liquid crystalline phases are typically found at detergent concentrations greater than about 30%. Inserted in the liquid crystalline areas are schemes of the liquid crystalline aggregates. The phase separation boundary for the system without PEG Is labelled a. The phase separation boundary downshifted by PEG with its detergent-rich phase in the lamellar crystalline area Is labelled b (Welte and Wacker 1991)

Fig. 3.1. Schematic phase diagram for non-ionic detergent water system. The abscissa and ordinate are without scale. Liquid crystalline phases are typically found at detergent concentrations greater than about 30%. Inserted in the liquid crystalline areas are schemes of the liquid crystalline aggregates. The phase separation boundary for the system without PEG Is labelled a. The phase separation boundary downshifted by PEG with its detergent-rich phase in the lamellar crystalline area Is labelled b (Welte and Wacker 1991)

Fig. 3.2. Illustration of the phase separation above the consolution boundary. A photosyn-thetic complex from a purple bacteria solubilized with dodecyl-maltoside is crystallized in the presence of PEG3350 (from C. Jungas, CEA-DEVM-Cadarache and E. Pebay-Peyroula). (a) Below solubility curve and consolution boundary, (b) Above consolution boundary, (c) Crystal nucleation within the droplets that are highly concentrated in protein and detergent, (d) The crystal growth depletes the protein droplets around the crystals, (e) and (f) The best crystals are obtained in thevicinity ofthe consolution boundary

Fig. 3.2. Illustration of the phase separation above the consolution boundary. A photosyn-thetic complex from a purple bacteria solubilized with dodecyl-maltoside is crystallized in the presence of PEG3350 (from C. Jungas, CEA-DEVM-Cadarache and E. Pebay-Peyroula). (a) Below solubility curve and consolution boundary, (b) Above consolution boundary, (c) Crystal nucleation within the droplets that are highly concentrated in protein and detergent, (d) The crystal growth depletes the protein droplets around the crystals, (e) and (f) The best crystals are obtained in thevicinity ofthe consolution boundary rich in detergent and also enhance the probability of protein-protein interactions to occur.

Figure 3.2 illustrates this process with a coloured protein, a bacterial reaction centre. The best crystals are often obtained just below the consolution boundary as illustrated in Fig. 3.2(f). The choice of the detergent is crucial not only for the solubilization of the protein but also for crystal formation. This choice was extensively discussed in several reviews (Reiss-Husson and Picot 1999); the favourite molecules can be deduced from crystallization conditions successful for membrane proteins (see statistics in http://www.mpibp-frankfurt.mpg.de/michel/pub-lic/memprotstruct.html). Neutron diffraction also showed that although crystal contacts imply proteins, the size of the detergent belt is an important parameter; it should not prevent protein-protein interactions from taking place and micelles have to fit well in empty spaces within the crystal packing. Additives such as hep-tane-triol influence the micelle radius and in some cases might favour better crystal contacts (Deisenhofer et al. 1985). The crystal contacts can also be favoured by enlarging the hydrophilic protein surface with an antibody fragment (Hunte and Michel 2003).

Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment