Solubilization of membrane proteins

It is emphasized that revealing the dynamics as well as the structure (or conformation) based on several types of spin-relaxation times is undoubtedly a unique and indispensable means, only available from NMR techniques at ambient temperature of physiological significance. Usually, the structure data themselves are available also from X-ray diffraction studies in a more refined manner. Indeed, better structural data can be obtained at lower temperature by preventing the unnecessary molecular fluctuations, which are major subjects in this chapter, since structural data can be seriously deteriorated for domains where dynamics are predominant even in the 2D or 3D crystalline state or proteoliposome at ambient temperature. It should be also taken into account that the solubilization of membrane proteins in detergents is an alternative means to study structure in solution NMR. However, it is not always able faithfully to mimick the biomembrane environment, because the interface structure is not always the same between the bilayer and detergent system. This typically occurs in the case of PLC-81(1-140) described in Section 4.2.4 and other types of peptide systems. 

Solubilization of Membrane Proteins. A modification of the procedure of Hjelmeland et al. (30) was employed. A 300-g portion of liquid-nitrogen frozen, 6-day Nicotiana silvestris cultured cells was suspended in 200 ml of 50 mM N-(2-hydroxyethyl)-piperazine-N -3-propanesulfonic acid (EPPS-KOH) buffer, 1 mM dithiothreitol (DTT), and 0.1 mM EDTA extraction buffer with constant stirring until completely suspended (20 min). The slurry was centrifuged in a Sorvall SS 34 rotor at 9,000g for 20 min at 4°C. The supernatant was passed through miracloth (Calbiochem). An aliquot... 

The use of purified proteins facilitates SPR determinations. Experiments with membrane proteins offer additional complexity, as their expression and purification can alter conformation, stability and/or function outside the cell environment. Solubilization of membrane proteins solves the problem in some cases, although detergents must be selected carefully (le Maire,... 

The choice of detergent used for solubilization of membrane proteins depends on both the protein and the subsequent method of purification. Some detergents with applications in membrane protein purification are listed in Table 5. 

Table 5 Detergents Used for Solubilization of Membrane Proteins...

Table 10.4 Detergent/protein (w/v) ratios for 90% maximal solubilization of membrane protein...

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