Expression of plant chaperonin-60 genes in Escherichia coli.

We have examined the expression in Escherichia coli of genes encoding a plant chloroplast molecular chaperone, chaperonin-60. Purified plant chaperonin-60 is distinct in that it contains two polypeptides, p60cpn-60 alpha and p60cpn-60 beta, which have divergent amino acid sequences (Hemmingsen, S. M., and Ellis, R. J. (1986) Plant Physiol. 80, 269-276; Martel, R., Cloney, L. P., Pelcher, L. E., and Hemmingsen, S. M. (1990) Gene (Amst.) 94, 181-187). The precise polypeptide composition(s) of the active tetradecameric specie(s) (cpn60(14)) has not been determined. Genes encoding the mature forms of the Brassica napus chaperonin polypeptides have been expressed separately and in combination in E. coli to produce three novel strains: alpha, beta, and alpha beta. The plant cpn60 polypeptides accumulated in soluble forms and to similar high levels in each. There was no conclusive evidence that p60cpn-60 alpha assembled into cpn60(14) species in alpha cells. In beta and alpha beta cells, the plant gene products assembled efficiently into cpn60(14) species. Thus, the assembly of p60cpn-60 alpha required the presence of p60cpn-60 beta, whereas the assembly of p60cpn-60 beta could occur in the absence of p60cpn-60 alpha. Significant proportions of the endogenous groEL polypeptides were not assembled into tetradecameric groEL14 in beta and alpha beta cells. Analysis of the tetradecameric species that did form indicated the presence of novel hybrid cpn6014 species that contained both plant and bacterial cpn60 polypeptides

Assessment of plant chaperonin-60 gene function in Escherichia coli.

Brassica napus chaperonin-60 alpha and chaperonin-60 beta genes expressed separately and in combination produce three novel Escherichia coli strains: alpha, beta, and alpha beta. In beta and alpha beta cells, the plant gene products assemble efficiently into tetradecameric cpn60(14) species, including novel hybrids containing both bacterial and plant gene products. The levels of authentic groEL14 are reduced in these cells (Cloney, L. P., Wu, H. B., and Hemmingsen, S. M. (1992) J. Biol. Chem. 267, 23327-23332). The assembly of cyanobacterial ribulose-P2 carboxylase (rubisco) in E. coli requires the activities of the endogenous chaperonin proteins. Furthermore, the extent to which assembly occurs is limited by the normal levels of expression of the groE operon (Goloubinoff, P., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 337, 44-47). We have now monitored the accumulation of cyanobacterial rubisco in E. coli alpha, beta, and alpha beta cells to assess the activity of the plant cpn60 gene products and effects on endogenous chaperonin functions. Expression of cpn-60 alpha alone did not enhance rubisco assembly, which is consistent with our previous observation that p60cpn-60 alpha required the presence of p60cpn-60 beta for assembly into cpn60(14) species. In contrast, expression of cpn-60 beta alone resulted in markedly enhanced rubisco assembly in cells that accumulated normal levels of both endogenous chaperonin polypeptides (groEL and groES). This demonstrates that assembled p60cpn-60 beta is functional as a chaperonin in E. coli. Co-expression of cpn-60 alpha and cpn-60 beta in cells with normal levels of expression of groES and groEL suppressed rubisco assembly. Increased expression of groES in cells in which cpn-60 alpha and cpn-60 beta were co-expressed relieved this suppression and resulted in enhanced rubisco assembly. Implications with respect to dependence of chloroplast cpn60 function on cpn10 are discussed

Amyloid-ß and a-Synuclein Decrease the Level of Metal-Catalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing

The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-ß (Aß) and a-synuclein (aS), associated with Alzheimer''s and Parkinson''s diseases, respectively, on the Cu2+-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu2+ is bound to Aß or aS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aß or aS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu2+ and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures

Structure of metal site in azurin, met 121 mutants of azurin, and stellacyanin investigated by 111m Cd Perturbed Angular Correlation (PAC)

The geometries of the metal sites in cadmium-substituted azurins have been investigated by Cd-111m perturbed angular correlation (PAC), The study includes wild type azurin as well as Met(121) mutants of azurin, where methionine has been substituted by Ala, Asn, Asp, Gin, Glu, and Leu.The nuclear quadrupole interaction of wild type azurin analyzed in the angular overlap model is well described as coordination of His(46), His(117), and Cys(112) and cannot be described by coordination of Met(121) and/or Gly(45).For most of the mutants, there exist two coordination geometries of the cadmium ion, With the exception of the Gau and Asp mutants, one of the conformations is similar to the wild type conformation. The other coordination geometries are either best described by a coordinating water molecule close to the original methionine position or by coordination by the substituting amino acid, These experiments show that even though the methionine does not coordinate it plays an important role for the geometry of the metal site.The nuclear quadrupole interaction of stellacyanin was also measured, The value resembles the most prominent nuclear quadrupole interaction of the Met(121) --> Gin mutant of Alcaligenes denitrificans azurin, indicating that the structures of the two metal sites are similar.Macromolecular Biochemistr