Membrane proteins solubilized integral

Several of the synthetic detergents used for dissolving membranes and solubilizing integral membrane proteins. Triton X-100 and octylglucoside are nonionic detergents cetyltrimethylammonium bromide and sodium dodecylsulfate (SDS) are ionic. SDS is also an effective denaturant of proteins and is used in polyacrylamide-gel electrophoresis (see chapter 6). 

Integral proteins are dissolved into the lipid bilayer of the membrane through interactions of the hydrophobic amino acid side chains and fatty acyl groups of phospholipids. In order to remove integral membrane proteins, the membrane must be disrupted by addition of detergents or other chaotropic reagents to solubilize the protein and to prevent aggregation and precipitation of the hydrophobic proteins upon their removal from the membrane. 

Much more is known about the interaction of cells with laminin. A high-affinity (Ad = 1 x 10 9 to 4 x 10-9) receptor for laminin has been found on many cells including myoblasts, tumor cells, and macrophages (Lesot et al., 1983 Rao et al., 1983 Malinoff and Wicha, 1983). The laminin receptor (Mr 67K) is solubilized by detergent and has all the characteristics of an integral membrane protein. A partial amino acid sequence of the receptor has been deduced from the nucleotide sequence of cDNA clones. These show a possible transmembrane sequence and suggest that this receptor has substantial cytoplasmic and extracellular domains (Wewer et al., 1986). 

Principle In this procedure erythrocytes are treated with Triton X-100 which is reported to solubilize the membrane lipid leaving the underlying cytoskeletal network intact. The cyto-skeletons are separated from cytosolic components, Triton and solubilized lipid by centrifugation through a sucrose solution. The high salt concentration of the sucrose solution ensures the removal of residual lipid and integral membrane proteins from the cytoskeletal network. 

More difficult, but also proniising to be honoured with success proves the isolation of pure, integral membrane proteins and their structure analysis by diffraction techniques. One avenue is given by defined solubilization with detergents and the evaluation of the small-angle (particle) scattering pattern from dilute solution (for reviews on this method, see Refs. and ). This has so far been attempted with bovine rhodopsin, the major protein component of retinal rod outer sement membranes with the Ca -dependent ATPase from sarcoplasmic reticulum... 

We simultaneously incorporate both lipid and protein by using dialysis to remove detergent from a solubilized lipid-protein mixture in the presence of the alkylsilanated substrate. Under our conditions, from the evidence in this paper and elsewhere (9), the surface structures appear to be single bilayer membranes. Our hypothesis is that the hydrocarbon chains attached to the surface serve as initiation sites for a lipid bilayer membrane to form as the detergent is slowly removed. The model is of a membrane that is anchored to the surface by hydrophobic interactions with the surface-bound hydrocarbon layer. Integral membrane proteins are retained in these structures by their interaction with the hydrophobic core of the membrane without being directly attached to the electrode surface. 

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. 

While micelles and bicelles have served as the membrane-mimetic solvent for the majority of solution NMR studies on integral membrane proteins, the identification of suitable NMR-compatible solvents remains a formidable challenge. With <30 unique multi-spanning membrane protein structures determined by solution NMR, there is a clear need to expand the current library of solubilization agents for this purpose. In response to this problem, creative approaches are currently being developed that may open the door to solution NMR for a wider range of membrane proteins in the future. Some of the systems that have started to yield promising results include nanodiscs, amphipols, and reverse micelles. 

Nanodiscs are comprised of a bilayer containing 130-160 lipids, maintained in a discrete, water-soluble state by the association of two copies of the membrane scaffold protein (MSP) from apolipoprotein A-I wrapped around the hydrophobic rim of the bilayer [199-202] (Fig. 3). Originally developed for the solubilization of functionally active integral membrane proteins, they have since been used for solid-state [203], and more recently, solution NMR applications [153, 204-207]. 

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