Protein incorporation

One of the best-studied examples of protein incorporation is the mitochondrial outer membrane. In yeast and Neurospora crassa none of the outer membrane proteins studied thus far are made as larger precursors. All of these proteins are made on free ribosomes and are incorporated into the outer membrane post-translationally [35,69,99]. The import of the porin (a pore-forming protein) is time and temperature dependent but does not require energy [35,99]. The incorporation of porin in vitro was found to be membrane specific [35]. How is this membrane specificity determined, and what anchors the outer membrane protein to the outer membrane  

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FIG. 22-86 Process scheme for protein extraction in aqueous two-phase systems for the downstream processing of intracellular proteins, incorporating PEG and salt recycling. RepHnted from Kelly and Hatton in Stephanopoulos (ed), op. cit. adapted from Qre-oe and Kula, op. cit.]... 

The Rieske protein II (SoxF) from Sulfolobus acidocaldarius, which is part, not of a bci or b f complex, but of the SoxM oxidase complex 18), could be expressed in E. coli, both in a full-length form containing the membrane anchor and in truncated water-soluble forms 111). In contrast to the results reported for the Rieske protein from Rhodobacter sphaeroides, the Rieske cluster was more efficiently inserted into the truncated soluble forms of the protein. Incorporation of the cluster was increased threefold when the E. coli cells were subject to a heat shock (42°C for 30 min) before induction of the expression of the Rieske protein, indicating that chaperonins facilitate the correct folding of the soluble form of SoxF. The iron content of the purified soluble SoxF variant was calculated as 1.5 mol Fe/mol protein the cluster showed g values very close to those observed in the SoxM complex and a redox potential of E° = +375 mV 111). 

The artificial lipid bilayer is often prepared via the vesicle-fusion method [8]. In the vesicle fusion process, immersing a solid substrate in a vesicle dispersion solution induces adsorption and rupture of the vesicles on the substrate, which yields a planar and continuous lipid bilayer structure (Figure 13.1) [9]. The Langmuir-Blodgett transfer process is also a useful method [10]. These artificial lipid bilayers can support various biomolecules [11-16]. However, we have to take care because some transmembrane proteins incorporated in these artificial lipid bilayers interact directly with the substrate surface due to a lack of sufficient space between the bilayer and the substrate. This alters the native properties of the proteins and prohibits free diffusion in the lipid bilayer [17[. To avoid this undesirable situation, polymer-supported bilayers [7, 18, 19] or tethered bilayers [20, 21] are used. 

Thus structural background suggests that the bound cyt c ccp adduct may actually consist of a distribution of structures. In this section, we consider the thermodynamics of binding cyt c and ccp, both for the native proteins, from different species, and proteins incorporating single site replacements, as prepared by site directed mutagenesis. 

Mimms LT, Zampighi G, Nozaki Y, Tanford C, Reynolds JA. Phospholipid vesicle formation and transmembrane protein incorporation using octyl gluco-side. Biochemistry 1981 20 833. 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

Lower incidence of heart disease has also been reported in populations consuming large amounts of soy products. Lowering of cholesterol is probably the best-documented cardioprotective effect of soy. ° Soy protein incorporated into a low-fat diet can reduce cholesterol and LDL-cholesterol concentrations and the soy isoflavones are likely to contribute to these effects. Soy isoflavones have been reported to improve cardiovascular risk factors in peripubertal rhesus monkeys, and inflammatory markers in atherosclerotic, ovariecto-mized monkeys. The potential role of phytoestrogens, including isoflavonoids, as cardioprotective agents has been extensively reviewed." ... 

Among these are the well-known E. coli leader peptidase355 356 and other signal peptidases.357 These are integral membrane proteins that cleave N-terminal signal sequences from proteins incorporated into plasma membranes. Another enzyme of this class is the lexA repressor and protease discussed in Chapter 28. 

Liposomes carrying membrane proteins incorporated via different reconstitution methods (detergent dialysis, sonication etc.)... 

Fig. 5.14 Helical proteins incorporating a crown ether amino acid analogue a view down the a-helix (left) and along the channel (top) [43]...

An area of research of obvious importance and immense scope involves the incorporation of amino acid analogues into proteins. Incorporation is accomplished either by chemical synthesis or by biochemical synthesis using the protein assembly mechanisms of nature. Halogenated, especially fluorinated, amino acid analogues have been particularly useful tools in this research. In this section, selected examples of research using such analogues will be described to illustrate strategies and principles employed. 

The mode of administration should be the same as that used clinically. In case of drug substances (e.g. proteins) incorporated into polymers, the drug substances is injected sub-cutaneously. However, special care has to be taken with regard to the skin area. If the drug is administered continuously to the same site, e.g. the neck, chronic local irritation / inflammation can induce sarcomas at the site of administration. To avoid this, the site of administration should be changed day by day (see also subacute to chronic toxicity) (Stammberger etal. 2002). 

Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., and Reynolds, J. A. (1981), Phospholipid vesicles formation and transmembrane protein incorporation using octyl glu-coside, Biochemistry, 20,833-840. 

Sloat, B. R., and Zhengrong, C. (2006), Strong mucosal and systemic immunities induced by nasal immunization with anthrax protective antigen protein incorporated in lipo-some-protamine-DNA particles, Pharm. Res., 23,262-269. 

Gochman-Hecht, H., and Bianco-Peled, H. (2006), Structure modifications of AOT reverse micelles due to protein incorporation, J. Coll. Interf. Sci., 297(1), 276-283. 

Carnitine is synthesized from lysine and methionine by the pathway shown in Figure 14.2 (Vaz and Wanders, 2002). The synthesis of carnitine involves the stepwise methylation of a protein-incorporated lysine residue at the expense of methionine to yield a trimethyllysine residue. Free trimethyllysine is then released by proteolysis. It is not clear whether there is a specific precursor protein for carnitine synthesis, because trimethyllysine occurs in a number of proteins, including actin, calmodulin, cytochrome c, histones, and myosin. 

The main metabolic function of vitamin K is as the coenzyme in the carboxyla-tion of protein-incorporated gluteimate residues to yield / -ceirboxygluteLmate -a unique type of ceirboxylation reaction, clearly distinct from the biotin-dependent carboxylation reactions (Section 11.2.1). 

Edelstein, R.L., and Distefano, M.D. (1997). Photoaffinity labeling of yeast farnesyl protein transferase and enzymatic synthesis of a Ras protein incorporating a photoactive isoprenoid. Biochem Biophys Res Commun 235 377-382. 

Fig. 3. Domain structure of the 303-amino acid-long P22 scaffolding protein as determined by mutational analysis. The N-terminal one-third of the protein is involved in posttranscriptional autoregulation of synthesis. An oligomerization domain, involved in dimerization and tetramerization, whose boundaries are not well defined is located in the central region of the protein. Mutations that ablate portal and minor protein incorporation are located in the region between residues 210 and 250, and the C-terminal region forms a tetratricopeptide-like fold that interacts with the coat protein.

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