Membrane proteins solubility

C. Tribet, R. Audebert, J.-L. Popot (1996) Amphipols polymers that keep membrane proteins soluble in aqueous solutions. Proc. Natl. Acad. Sci. USA, 93 15047-15050... 

C. Prata, F. Giusti, Y. Gohon, B. Pucci, J.-L. Popot, C. Tribet (2001) Non-ionic amphiphilic polymers derived from Tris(hydroxymethyl)-acrylamidomethane keep membrane proteins soluble and native in the absence of detergent. Biopolymers, 56 77-84... 

The presence of non-ionic detergent avoided denaturing conditions and interference with purification steps whilst maintaining membrane protein solubility. 

Problems Serva Blue G presumably prefers to attach to trans-membrane regions. Large membrane proteins thus show a lesser charge density than small membrane proteins. Soluble proteins bind still less stain. The charge-to-mass ratio of different protein-stain complexes is thus not constant, and the native electrophoresis does not separate the protein complexes by MW (personal communication by A. Schrattenholz, Mainz). Soluble marker proteins such as thyroglobulin, ferritin, and the like smudge in the gel or partially disintegrate into subimits. 

Formation of vesicles is likely to require interaction with the soluble coat components of cytoplasmic domains of certain integral membrane proteins that may serve as... 

Mitochondrial permeability transition involves the opening of a larger channel in the inner mitochondrial membrane leading to free radical generation, release of calcium into the cytosol and caspase activation. These alterations in mitochondrial permeability lead eventually to disruption of the respiratory chain and dqDletion of ATP. This in turn leads to release of soluble intramito-chondrial membrane proteins such as cytochrome C and apoptosis-inducing factor, which results in apoptosis. 

Once the proteins have passed the quality control system of the early secretory pathway, they are transported in vesicles via the individual compartments of the Golgi apparatus to the plasma membrane. Soluble proteins are transported in the vesicle lumen, membrane proteins are integrated in the vesicle membrane. The transport to the cell surface is the default pathway for secretory and membrane proteins. Proteins may also become part of one of the intracellular compartments along the secretory pathway, but only if they contain specific retention signals. 

Rieske proteins are constituents of the be complexes that are hydro-quinone-oxidizing multisubunit membrane proteins. All be complexes, that is, bci complexes in mitochondria and bacteria, b f complexes in chloroplasts, and corresponding complexes in menaquinone-oxidizing bacteria, contain three subunits cytochrome b (cytochrome 6e in b f complexes), cytochrome Ci (cytochrome f in b(,f complexes), and the Rieske iron sulfur protein. Cytochrome 6 is a membrane protein, whereas the Rieske protein, cytochrome Ci, and cytochrome f consist of water-soluble catalytic domains that are bound to cytochrome b through a membrane anchor. In Rieske proteins, the membrane anchor can be identified as an N-terminal hydrophobic sequence (13). 

Scientists initially approached structure-function relationships in proteins by separating them into classes based upon properties such as solubility, shape, or the presence of nonprotein groups. For example, the proteins that can be extracted from cells using solutions at physiologic pH and ionic strength are classified as soluble. Extraction of integral membrane proteins requires dissolution of the membrane with detergents. 

The number of different proteins in a membrane varies from less than a dozen in the sarcoplasmic reticulum to over 100 in the plasma membrane. Most membrane proteins can be separated from one another using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique that has revolutionized their study. In the absence of SDS, few membrane proteins would remain soluble during electrophoresis. Proteins are the major functional molecules of membranes and consist of enzymes, pumps and channels, structural components, antigens (eg, for histocompatibility), and receptors for various molecules. Because every membrane possesses a different complement of proteins, there is no such thing as a typical membrane structure. The enzymatic properties of several different membranes are shown in Table 41-2. 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Figure 4. Purification of PemB from E. coli K38 pGPl-2/pPME6-5 cells. Proteins were separated by urea-SDS-PAGE. Lane 1, induced cell lysate lane 2, soluble protein fraction from induced cells lane 3, membrane fraction from non-induced cells lane 4, membrane fraction from induced cells lane 5, membrane proteins not extracted by Triton X-100 lane 6, membrane proteins extracted by Triton X-100 lane 7, PemB purified by preparative electrophoresis. The molecular weight standard positions are indicated.

The molecular weights of all SERCA-type Ca " transport ATPases are in the range of 100-110 kDa. Their N-terminal sequences are similar Met-Glu-X(Ala, Asn, Glu, Asp)-X (Ala, Gly, He). The Met-Glu-X-X sequence serves as a signal for the acetylation of N-terminal methionine both in soluble and in membrane proteins [71,72]. 

Cierpicki, T., Bushweller, J. H. Charged gels as orienting media for measurement of residual dipolar couplings in soluble and integral membrane proteins. J. Am. Chem. Soc. 2004, 126, 16259-16266. 

In the case of PS II membrane proteins, as discussed above, the hydrophobic and hydrophilic pairs of attached lipids can partially support the protein complex at the air-water interface, despite their large size and density. However, in the case of PS II core complex, the detergent strips the attached lipids and some extrinsic proteins. The remaining protein complex is water soluble. It is very difficult to prepare a stable monolayer of water-soluble proteins with the Langmuir method. Indeed, it is hard to directly prepare a stable monolayer of PS II core complex because of its water solubility as well as density. One possible solution is to change the density and ionic strength of the subphase [9]. 

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