Halide binding by the purified halorhodopsin chromoprotein. II. New chloride-binding sites revealed by 35Cl NMR

Halorhodopsin is a light-driven chloride pump in the cell membrane of Halobacterium halobium. Recently, a polypeptide of apparent Mr = 20,000 has been purified that contains the halorhodopsin chromophore. Here we use 35Cl NMR to show that the purified chromoprotein possesses two previously unknown classes of chloride-binding sites. One class exhibits a low affinity (KD much greater than 1 M) for chloride and bromide. The second class exhibits a higher affinity (KD = 110 ± 50 mM) for chloride and also binds other anions according to the affinity series I-, SCN- greater than Br-, NO-3 greater than Cl- greater than F- , citrate. Both classes of NMR site remain intact at pH 11, indicating that the essential positive charges are provided by arginine. Also, both classes are unaffected by bleaching, suggesting that the sites are not in the immediate vicinity of the halorhodopsin chromophore. Although the chromoprotein also appears to contain the chloride- transport site (Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi, J. K. (1984) J. Biol. Chem. 259, 2179-2184), this site was not detected by 35Cl NMR, suggesting that the transport site is in the interior of the protein where it is sampled slowly by chloride in the medium. It is proposed that the purified chromoprotein possesses a channel leading from the medium to the transport site and that the channel contains the high affinity NMR site which facilitates the migration of chloride between the medium and the transport site. We have also used 35Cl NMR to study chloride binding to purified monomeric bacteriorhodopsin; however, this protein contains no detectable chloride-binding sites

Activation of the phosphosignaling protein CheY. I. Analysis of the phosphorylated conformation by 19F NMR and protein engineering

CheY, the 14-kDa response regulator protein of the Escherichia coli chemotaxis pathway, is activated by phosphorylation of Asp57. In order to probe the structural changes associated with activation, an approach which combines 19F NMR, protein engineering, and the known crystal structure of one conformer has been utilized. This first of two papers examines the effects of Mg(II) binding and phosphorylation on the conformation of CheY. The molecule was selectively labeled at its six phenylalanine positions by incorporation of 4-fluorophenylalanine, which yielded no significant effect on activity. One of these 19F probe positions monitored the vicinity of Lys109, which forms a salt bridge to Asp57 in the apoprotein and has been proposed to act as a structural "switch" in activation. 19F NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered local structural changes in the activation site, but did not perturb the probe of the Lys109 region. The structural changes associated with phosphorylation were then examined, utilizing acetyl phosphate to chemically generate phsopho-CheY during NMR acquisition. Phosphorylation triggered a long-range conformational change extending from the activation site to a cluster of 4 phenylalanine residues at the other end of the molecule. However, phosphorylation did not perturb the probe of Lys109. The observed phosphorylated conformer is proposed to be the first step in the activation of CheY; later steps appear to perturb Lys109, as evidenced in the following paper. Together these results may give insight into the activation of other prokaryotic response regulators

Solitons in the one-dimensional forest fire model

Fires in the one-dimensional Bak-Chen-Tang forest fire model propagate as solitons, resembling shocks in Burgers turbulence. The branching of solitons, creating new fires, is balanced by the pair-wise annihilation of oppositely moving solitons. Two distinct, diverging length scales appear in the limit where the growth rate of trees, $p$, vanishes. The width of the solitons, $w$, diverges as a power law, $1/p$, while the average distance between solitons diverges much faster as $d \sim \exp({\pi}^2/12p)$.Comment: 4 pages with 2 figures include

The Determinants Of Human Wellbeing In Professional Activities

The article discusses the issue of person's attitude towards success in his professional activities, which are the value of human labor and his wellbeing in professioanl activity. Personal attitude and substantial components of socio-psychological factors of attitude development provides the field of welfare of the employee. It focuses on the implementation of efficient technologies of formation attitude towards success in the process of training and retraining. The object of the research was the OJSC Evrazruda candidate pool members studying at the Evraz Siberia Regional Staff Training Center, as well as a group of experts (engineer, metallurgists). The techniques of V. K. Gerbachevsky, V. V. Stolin, S. R. Panteleyev, H. J. Eysenck, M. Rokeach, N. M. Peysakhov and J. Rotter were used in the research. The study showed the interconnection of a person's attitude with the dominant features of the component structure of motivational, volitional and self-consciousness factors. They are the determinants of wellbeing and quality of life in professional activity

Effects of thermal fluctuation and the receptor-receptor interaction in bacterial chemotactic signalling and adaptation

Bacterial chemotaxis is controlled by the conformational changes of the receptors, in response to the change of the ambient chemical concentration. In a statistical mechanical approach, the signalling due to the conformational changes is a thermodynamic average quantity, dependent on the temperature and the total energy of the system, including both ligand-receptor interaction and receptor-receptor interaction. This physical theory suggests to biology a new understanding of cooperation in ligand binding and receptor signalling problems. How much experimental support of this approach can be obtained from the currently available data? What are the parameter values? What is the practical information for experiments? Here we make comparisons between the theory and recent experimental results. Although currently comparisons can only be semi-quantitative or qualitative, consistency is clearly shown. The theory also helps to sort a variety of data.Comment: 26 pages, revtex. Journal version. Analysis on another set of data on adaptation time is adde