Conformational changes of the betaine transporter BetP from Corynebacterium glutamicum studied by pulse EPR spectroscopy.

The betaine transporter BetP from Corynebacterium glutamicum is activated by hyperosmotic stress critically depending on the presence and integrity of its sensory C-terminal domain. The conformational properties of the trimeric BetP reconstituted in liposomes in the inactive state and during osmotic activation were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Comparison of intra- and intermolecular inter spin distance distributions obtained by double electron-electron resonance (DEER) EPR with the crystal structure of BetP by means of a rotamer library analysis suggest a rotation of BetP protomers within the trimer by about 15° as compared to the X-ray structure. Furthermore, we observed conformational changes upon activation of BetP, which are reflected in changes of the distances between positions 545 and 589 of different protomers in the trimer. Introduction of proline at positions 550 and 572, both leading to BetP variants with a permanent (low level) transport activity, caused changes of the DEER data similar to those observed for the activated and inactivated state, respectively. This indicates that not only displacements of the C-terminal domain in general but also concomitant interactions of its primary structure with surrounding protein domains and/or lipids are crucial for the activity regulation of BetP.

Human alpha2-macroglobulin: genotype-phenotype relation.

A pentanucleotide deletion polymorphism in the gene of alpha2-macrolgobulin (alpha2-M) is suggested to be associated with late-onset Alzheimer's disease (AD), though controversial results have been reported. The underlying assumption is that the intronic pentanucleotide deletion may affect the biological function and quantity of the inhibitor and thus contribute to the AD pathology. In the present study we have analyzed the distribution of the deletion polymorphism within a group of 227 healthy Caucasians. In parallel studies, we determined the plasma concentrations of total and transformed alpha2-M. A strong correlation of the total concentration of alpha2-M with age was ascertained (r(s) = -0.54, P < 0.001). However, no significant correlation between age and the genotypes (P = 0.68) was detected, and no statistically significant effect of the genotype on the concentrations of total and transformed alpha2-M was found (P = 0.49 and 0.96, respectively). A significant correlation was observed between total and transformed alpha2-M in the genotype groups Ins/Ins (r(s) = 0.56, P < 0.001) and Ins/Del (r(s) = 0.35, P < 0.004). Furthermore, in the entire data set, a significantly elevated concentration of total alpha2-M was found in females as compared to males (P = 0.003). There was a slight but nonsignificant difference in the genotype distributions between males and females (P = 0.14). To test the proposed existence of genotype-specific alterations of functional properties of alpha2-M, we isolated alpha2-M from the plasma of carriers with different genetic background and analyzed the alpha2-M subunit structure as well as the binding of the inhibitor to growth factors/cytokines, to amyloid-beta and to the receptor. The experiments failed to reveal any genotype-specific functional alterations of the alpha2-M. The absence of abnormalities in alpha2-M mRNA and protein suggests that the alpha2-M deletion polymorphism is probably not associated with functional deficiencies important in AD pathology. However, it can be speculated that the observed general age-related alpha2-M deficiency may lead to accelerated accumulation of amyloid-beta, which might be relevant to AD pathology.

Expression of two nblA-homologous genes is required for phycobilisome degradation in nitrogen-starved Synechocystis sp. PCC6803.

One of the responses exhibited by cyanobacteria when they are limited for an essential nutrient is the rapid degradation of their light-harvesting complex, the phycobilisome. Phycobilisome degradation is an ordered proteolytic process, visible by a color change of the cyanobacterial cell from blue-green to yellow-green (chlorosis). The small polypeptide NblA plays a key role in degradation of phycobilisomes in Synechococcus sp. PCC7942. Unlike Synechococcus, Synechocystis sp. PCC6803 has two nblA-homologous genes, nblA1 and nblA2, which are contiguous on the genome. Here we show that nblA1 and nblA2 are simultaneously expressed in Synechocystis 6803 upon nitrogen deprivation, and are both required for phycobilisome degradation.

Evidence for propeptide-assisted folding of the calcium-dependent protease of the cyanobacterium Anabaena.

The Ca(2+)-dependent protease of the cyanobacterium Anabaena variabilis is a cytoplasmic enzyme with a substrate specificity like trypsin. Its previously published DNA sequence [Maldener, I., Lockau, W., Cai, Y. & Wolk, C. P. (1991) Mol. Gen. Genet. 225, 113-120] contained a sequencing error. Here we report the corrected sequence which shows, that the Ca(2+)-protease belongs to the family of subtilases (subtilisin-like serine proteases). Consistent with its cytoplasmic localization, a pre-sequence is not found. The enzyme is produced as a precursor with a large amino-terminal propeptide. Expression of the pro-region and mature region (protease domain) in Escherichia coli cells in trans demonstrates that formation of the active enzyme requires the propeptide. The results demonstrate that propeptide-assisted protein folding also occurs with cytoplasmic enzymes, in support of the hypothesis that this mechanism is a widespread phenomenon.