Fragments, proteins that interact with membranes

In be complexes bci complexes of mitochondria and bacteria and b f complexes of chloroplasts), the catalytic domain of the Rieske protein corresponding to the isolated water-soluble fragments that have been crystallized is anchored to the rest of the complex (in particular, cytochrome b) by a long (37 residues in bovine heart bci complex) transmembrane helix acting as a membrane anchor (41, 42). The great length of the transmembrane helix is due to the fact that the helix stretches across the bci complex dimer and that the catalytic domain of the Rieske protein is swapped between the monomers, that is, the transmembrane helix interacts with one monomer and the catalytic domain with the other monomer. The connection between the membrane anchor and the catalytic domain is formed by a 12-residue flexible linker that allows for movement of the catalytic domain during the turnover of the enzyme (Fig. 8a see Section VII). Three different positional states of the catalytic domain of the Rieske protein have been observed in different crystal forms (Fig. 8b) (41, 42) ... 

The firagments in the material to be analyzed (DNA, RNA, or protein) are separated by gel electrophoresis. The smaller molecules travel faster and appear nearer the bottom of the gd. The bands of material in the gel are transferred or blotted to the surface of a membrane. The membrane is incubated with a (usually radioactive) labeled probe that will specifically bind to the molecules of interest. Visualization of the labded probe (usually by autoradiography) will reveal which band interacted with the probe. The most common types of blots are compared in Table 1-7-1. Most typically, DNA restriction fragments are analyzed on a Southern blot. 

Figure 3 Presenilin, the y-secretase complex, and the proteolysis of APP to Ap. (a) Presenilin is processed into two pieces, an N-terminal fragment (NTF, dark portion) and a C-terminal fragment (CTF, light portion) that remain associated. Each fragment donates one aspartate that is essential for y-secretase activity. APP is cleaved first in the extracellular domain by p-secretase, and the remnant is cleaved twice within the membrane by y-secretase to produce the Ap peptide of Alzheimer s disease (secreted) and the intracellular domain (AlCD, freed into the cytosol), (b) Presenilin interacts with three other membrane proteins, nicastrin, Aph-1, and Pen-2, to form active y-secretase.

In principle any binding protein or receptor site or membrane fragment containing a receptor site can be purified by the use of an affinity medium containing the moiety with which the protein or receptor interacts. The extensive bibliography rapidly building up in this area of application indicates that, as with most other applications in affinity chromatography, the principle appears to be valid. 

Endocytosis may not be required for the entry of an invasive adenylate cyclase from Bordello pertussis (Hanski and Ferfel, 1985 Donovan and Storm, 1990). This is a single chain protein (mol. wt. approx. 200 kDa) which resembles the edema factor from anthrax toxin in that it must interact with calmodulin to become active. In contrast to anthrax toxin, it consists of only one polypeptide which is, however, easily cleaved by proteases and thereby activated. An enzymatically active 45 kDa fragment is not active on whole cells, but it could in conjunction with the rest of the molecule enter the cytosol. The facts that this toxin acts much more rapidly than anthrax toxin, and that it is active even at 4 °C and on erythrocytes that have little, if any, endocytosis, suggest that the toxin is able to penetrate directly through the cell surface membrane. 

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