Basic blue protein

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

Cucumber basic blue protein has been put in a class of its own in view of having a disulfide, unUke plastocyanin, although its fold is generally like that of plastocyanin. 

Cucumber basic blue protein (Cbp) is a protein without known function, also known as cusacyanin or plantacyanin. Its structure (Guss et al., 1988) completes the repertoire of cupredoxins with known structures. The topology of its folding is similar (Fig. 5) to those of plastocyanin and azurin, as might have been expected from sequence similarities and... 

Fig. 5. (a) Copper site in cucumber basic blue protein (Cbp). (b) Ribbon drawing of the Cbp backbone, (c and d) Schematic of Cbp topology. 

Basic blue proteins have been isolated from a number of plant sources and have previously been referred to as plantacyanin (Aikazyan and Nalbandyan, 1975,1979 Sakurai et al, 1982). The protein from cucumber has been the most extensively studied of the basic blue proteins and the crystal structure is now available (Guss et al., 1988). The function of this basic blue protein is unknown, however, it is probably not involved in photosynthetic electron transport as it will not replace plastocyanin in that electron transport chain (Adman, 1985). 

The cucumber basic blue protein is made up of a single polypeptide chain of 96 amino acids containing a single copper ion with a molecular weight of 10000 (Adman, 1985). The amino acid sequence (Bergman et al., 1977) is very similar to those of stellacyanin and umecyanin. The copper in cucumber blue protein displays the characteristic spectroscopic properties of a typical blue copper site (Table 5-5). 

The crystal structure of cucumber basic blue protein has now been refined to 3.0 A resolution (Adman, 1985). The protein consists of eight strands, only five of which form a P-sandwich and the protein has less P-sheet character than plastocyanin or azurin. The ligands to copper are provided by the side chains of His-39, Cys-79, His-84 and Met-89. The copper site has the N2SS coordination seen in plastocyanin. The imidazole rings of the His-39 and His-94 residues are exposed to the solvent providing a likely entry site for electon transfer to the copper centre. 

The type I copper sites function as electron transfer centers in the blue copper proteins and in multicopper enzymes, particularly oxidases (33). They are characterized by their intense blue color, their unusually small A values, and their very positive redox potentials (Table II). X-ray crystal structures of several blue copper proteins have been determined, notably plastocyanin (34), azurin (35), cucumber basic blue protein (36), and pseudoazurin (37). The active site structures show marked similarities but also distinct differences (Fig. 8). 

The physiological functions of the phytocyanins are also currently unknown. This family of proteins consists of stellacyanin (20 kD) [97, 101], umecyanin (14 kD), the basic blue protein (from cucumber = plantacyanin) (8 kD), and cusacyanin (molecular masses see [17]). 

The molecular structure of another blue protein, the phytocyanin (phytocyanins are electron carriers found in the non-photosynthetic part of plants) cucumber basic protein (FW=10 100), also known as plantacyanin, is shown in Figure 33.60... 

Guss, J. M., et al. (1988). Phase determination by multiple-wavelength x-ray diffraction crystal structure of a basic blue copper protein from cucumbers. Science 241, 806-811. 

Unknown stellacyanin, umecyanin, cucumber basic blue copper protein... 

Bond Cu(SCPh3)L Azurin Populus plastocyanin Enteromorpha prolifera plastocyanin Pseudoazurin Cucumber basic blue copper protein"... 

Upon perturbation of the geometry of the blue copper center, from plastocya-nin to cucumber basic blue to nitrite reductase (a green protein), flie Cys S n —>Cu CT intensity decreases while the Cys S cr—>Cu CT intensity increases... 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

A standard Lowry-based protein assay has been adjusted to the special conditions encountered with skin [126], Basically, proteins reduce an alkaline solution of Cu(II)-tartrate to Cu(I) in a concentration-dependent manner. Then, the formation of a blue complex between Folin-Ciocalteau reagent (a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids) and Cu(I) can be measured spectrophotometrically at 750 nm. A calibration curve can be obtained by dissolving known amounts of stratum corneum in 1 M sodium hydroxide. A piece of tape that has not been in contact with skin is subjected to an identical procedure and serves as negative control. The method was recently adapted to a 96-well plate format, notably reducing analysis times [129],... 

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

We have studied the blue copper proteins plastocyanin, azurin, cucumber basic protein ( ) and nitrite reductase (NiR) (159,160). We shah focus on these four in the remainder of this section. 

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