Solubilization of membrane

Helenius, A. Simons, K., Solubilization of membranes by detergents, Biochim. Biophys. Acta 413, 29-79 (1975). 

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. 

Subsequent to possible solubilization of membrane-bound proteins, solubilization must be verified. The criteria listed in Table 2 are relevant in assessing whether solubilization has been accomplished. To ascertain whether the solubilized protein has retained biological activity, membrane reconstitution (28) is attempted subsequent to detergent removal (24). Reconstitution is often visualized by electron microscopy employing either negative staining or freeze fracture. 

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 early work on the interactions of synthetic surfactants and fatty acid anions with proteins was reviewed by Putnam [9] in 1948 and the following 20 years by Steinhardt and Reynolds [10]. The expansion of the field has led more recently to reviews and monograph chapters dealing with specific areas of protein-surfactant interactions, such as the solubilization of membranes by detergents [11] and the use of surfactants in membrane solubilization and reconstitution of membrane proteins [12], as well as more general reviews [13,14,15],... 

The haemolytic activities of natural CyDs are reported to be in the order p- > a- > y-CyD. These differences are ascribed to the differential solubilization of membrane components by each CyD. When the CyD cavity is modified by chemical derivatization, its effects on cell membranes can be changed dramatically from those of parent CyDS. When the muscle tissue damage due to the injection of hydrophilic CyDs was compared with that of mannitol and nonionic surfactants, following a single injection (100 mg ml" ) of the compounds into M. vastus lateralis of rabbits (Fig. 38.2), a-CyD and DM-P-CyD showed a relatively high irritation reaction, the degree of... 

During the solubilization of membranes, the purification of integral membrane proteins, and the reconstitution of membranes, gentler detergents, such as octyl glucoside, are used in preference to sodium dodecyl sulfate (SDS). Explain why. 

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