Lipid to Protein Ratio

The reconstitution of membrane proteins into bilayers is achieved by mixing lipids and protein, both solubilized in detergents, and decreasing the detergent concentration. Figure 1 shows an example where the concentration of octyl-polyoxyethylene (8-POE) was decreased by dilution, and the formation of structures of different sizes was monitored using dynamic light scattering (7). The dilution experiment led to the formation of vesicles with egg phosphatidylcholine (EggPC) or vesicles and 2D crystals with EggPC and the porin OmpF. The latter assembled only if the dilution rate was slow. The relationship between detergent concentration and structure sizes can be described as the "3-stage" model of Lichtenberg et al. (30). Stage I (crystals/vesicles area) is characterized by a detergent concentration too low to disrupt the lipid bilayer. Stage II (intermediate structures area) is the region of detergent concentration where lipid bilayer and mixed micellar structures coexist. The micelle-bilayer transition region (Stage II) was found to be the key to reconstitution and, by implication, to 2D crystallization (7,11). Cryo-electron microscopy (9) has shown for several lipid—detergent systems that this transition involves the formation of worm-like extended lipid micelles, probably capped by detergents that must convert to vesicles on detergent removal. Such rod-like structures are therefore thought to be important intermediates in the formation of 2D crystals.

Figure 1. Hydrodynamic radii on dilution of a mixed micellar suspension containing a detergent (8-POE) and either lipid (EggPC) or lipid and a membrane protein (porin OmpF). Above 15 mM 8-POE, uniform micelles are seen. Below 15 mM 8-POE, each treatment produces a range of structure radii with large crystals seen in the presence of OmpF The dilution is represented as a function of detergent concentration to illustrate the 3-stage model. Large bilayer structures (crystals/vesicles) at low detergent concentration (Stage I). Small micelles at high detergent concentration (Stage III). Mixture of structures at intermediate detergent concentration (Stage II). Black arrows indicate the saturation points, and white arrows indicate the solubilization points. Data from Reference 7. Figure was modified from Reference 15.

Figure 1. Hydrodynamic radii on dilution of a mixed micellar suspension containing a detergent (8-POE) and either lipid (EggPC) or lipid and a membrane protein (porin OmpF). Above 15 mM 8-POE, uniform micelles are seen. Below 15 mM 8-POE, each treatment produces a range of structure radii with large crystals seen in the presence of OmpF The dilution is represented as a function of detergent concentration to illustrate the 3-stage model. Large bilayer structures (crystals/vesicles) at low detergent concentration (Stage I). Small micelles at high detergent concentration (Stage III). Mixture of structures at intermediate detergent concentration (Stage II). Black arrows indicate the saturation points, and white arrows indicate the solubilization points. Data from Reference 7. Figure was modified from Reference 15.

At the start of a typical reconstitution experiment, an excess of detergent ensures a homogenous distribution of protein and lipid in micelles. As detergent concentration is decreased, lipids and proteins interact due to the exposure of their hydrophobic surfaces. With an excess of lipid over protein, the protein is mainly incorporated into lipid bilayers, similar to its native state. In an excess of protein over lipid, the protein mostly ends up in amorphous aggregates, perhaps denatured. An important parameter is, therefore, the lipid:protein ratio (LPR), which should be low enough to promote crystal contacts between protein molecules, but not so low that the protein is lost to aggregation. When the membrane protein is reconstituted from a mixture of solubilized components, crystal ordering of proteins may occur during reconstitution. For crystal packing during reconstitution, the LPR of the reconstitution experiment must be as low as possible to ensure close packing without leading to excessive aggregation. While the lipid content of the reconstitution mixture is, in general, a well-controlled parameter, the content of monodisperse protein is sometimes unknown, because protein assays do not indicate the amount of aggregates.

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