Comparison of the transmembrane helices of bovine rhodopsin in the crystal structure and the C α template based on cryo-electron microscopy maps and sequence analysis of the G protein-coupled receptors

G protein-coupled receptors (GPCR) are activated by a diverse array of extracellular signals, ranging from light to polypeptide molecules. The receptors propagate these signals intracellularly using G protein secondary messenger pathways. A common feature in the architecture of these receptors is their seven transmembrane domains. The first crystal structure of a GPCR, bovine rhodopsin, has recently been solved at 2.8 Å. We compared the seven membrane-spanning helices (TMH) from the crystal structure of bovine rhodopsin with those from the low-resolution model of bovine rhodopsin based on the cryo-electron microscopy structure of frog rhodopsin developed by Dr Joyce Baldwin. The model developed by Baldwin used a consensus sequence approach to predict the rotational position of each helix with respect to the other six helices. Superposition of the entire helix bundle of the Baldwin model with the crystal structure gave a RMS difference (RMSD) of 3.2 Å for the 198 C f atoms which suggests a high level of similarity in the arrangement of the helices. Except for TMH IV (RMSD of 4.0 Å), the position of corresponding helices within the helix bundle overlapped well. The superposition of individual helices showed that the RMSD values over 3 Å in the global superposition were largely due to one or more of the following: (i) differences in the unraveling and kinks for these helices, (ii) translation of TMH perpendicular to the membrane and (iii) rotation of helices up to 31°, except for TMH IV in which an additional contribution to the RMSD came from the aforementioned observation. As other crystal structures of GPCRs become available, a comparison with the Baldwin consensus model may reveal larger differences than those observed here.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Crystallographic analysis of two site-directed mutants of Azotobacter vinelandii ferredoxin

The crystal structure of the C24A mutant of Azotobacter vinelandii 7Fe ferredoxin (FdI) has been solved and refined at 2.0-A resolution. The structure is isomorphous to native FdI except at the site of mutation where A24 moves toward the [4Fe-4S] cluster. In spite of this inefficient packing results: three of five van der Waals contacts from the S gamma of C24 in native FdI are lost and the remaining two become longer. Consequently, the [4Fe-4S] cluster is either disordered or has a higher temperature factor (B factor) compared to the rest of the C24A FdI molecule. In addition, the entire C24A FdI structure has a higher overall B factor than native FdI. Therefore, in comparison to native FdI, the C24A mutant is isomorphous but exhibits large differences in B factor, especially at the [4Fe-4S] cluster. In contrast, the C20A FdI structure (Martin, A. G., Burgess, B. K., Stout, C. D., Cash, V. L., Dean, D. R., Jensen, G. M., and Stephens, P. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 598-602), which contains large structural rearrangements in the vicinity of the [4Fe-4S] cluster, exhibits essentially no change in B factor. The conformational change observed at residue 24 is similar in both C24A and C20A FdI structures. The solvent accessibility of the Fe atoms in the [3Fe-4S] and [4Fe-4S] clusters is similar in C24A, C20A, and native FdI.     

The normal replication terminus of the Bacillus subtilis chromosome, terC, is dispensable for vegetative growth and sporulation

The Bacillus subtilis strains CU1693, CU1694 and CU1695 were shown by hybridization analysis to carry large deletions of the terminus region that originated within discrete fragments of the SP beta prophage genome. The absence of terC in CU1693 was demonstrated definitively by the identification of a novel junction fragment comprising SP beta DNA and DNA that lies on the other side of terC in the parent strain. This represented the deletion of approximately 230 kb of CU1693 DNA, with the removal of approximately 150 kb to the left of terC and approximately 80 kb to the right of terC. The lack of hybridization of CU1694 and CU1695 DNA to cloned DNA carrying the terC sequence and to cloned DNAs flanking terC suggested that terC is absent from the chromosome of each of these strains also, and that the deletions in CU1694 and CU1695 extend beyond the segment of the terminus region that has been mapped and cloned. The normal growth rate and morphology of CU1693, CU1694 and CU1695 relative to the parent strain when grown in complex medium indicated dispensability of terC for vegetative growth and division. B. subtilis SU153 was constructed using a specific deletion-insertion vector that was designed to effect the deletion of 11.2kb of DNA spanning terC, with the removal of approximately 9.7kb to the left of terC and approximately 1.kb to the right of terC. This manipulation did not introduce any readily detectable auxotrophic requirement. Physiological characterization of SU153 confirmed the dispensability of terC for vegetative growth and cell division, and also established the lack of requirement of terC for the specialized cell division that is associated with formation of the bacterial endospore.     

Uptake of l-Phenylalanine in Synchronous Chlorella fusca. Characterization of the Uptake System

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a K_m= 5 μM. V_max, was 0.1 nmol/min × 10⁷ cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l -phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l -phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L-phenylalanine. Variations in the ratio of Na4⁺ to K⁺ in the external solution during uptake experiments did not have any influence upon the uptake rate of l -phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4-Dinitro-phenol inhibited the uptake completely. Exchange between internal and external l -phenylalanine could not be demonstrated. The K_m value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l -phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells.     

Thiol-based redox signalling: rust never sleeps

Cysteine residues in proteins are covalently modified under conditions of oxidative and nitrosative stress by oxidation, nitrosation, glutathionylation and disulfide formation. Modifications induce conformational changes in substrate proteins, effecting signal cascades that evoke a biological response. A growing number of structures with modified cysteines are allowing a piecemeal understanding of the mechanistic aspects of these signalling pathways to emerge. Conformational changes upon conjugation of nitric oxide and glutathione are generally small and often accompanied by a local increase in protein disorder. Burial of nitric oxide is also apparent, which may increase the timeframe of signalling. Conformational changes upon disulfide formation/reduction range from the small to the spectacular. They include order/disorder transitions; oxidation of disulfides following expulsion of metals such as Zn; major reorganisation or "morphing" of portions of the polypeptide backbone; and changes in quaternary structure including domain swapping.     

Transport of free polymannose-type oligosaccharides from the endoplasmic reticulum into the cytosol is inhibited by mannosides and requires a thapsigargin-sensitive calcium store

The transport of free polymannose-type oligosaccharides from the lumen of the endoplasmic reticulum into the cytosol has been recently demonstrated (Moore,S.E.H., et al., 1995, EMBO J., 14, 6034-6042), but at present little is known of the characteristics of this process. Here, it is shown that inhibition of the transport of endogenously synthesized metabolically radiolabeled free oligosaccharides out of the endoplasmic reticulum into the cytosol of permeabilized HepG2 cells occurs when assays are conducted in the presence of mannose (IC50, 4.9 mM), or its derivatives modified at the first carbon (C1) of the sugar ring; alpha-methyl mannoside (IC50, 2.0 mM), mannoheptulose (IC50, 1.6 mM), and alpha-benzyl mannoside (IC50, 0.8 mM), whereas other monosaccharides (50 mM), differing from mannose at position; C2 (glucose), C3 (altrose), C4 (talose), C5 (l-rhamnose), and C6 (mannoheptose), have little effect. N-Acetylglucosamine does not inhibit oligosaccharide transport and, furthermore, although mannobioses and a mannotriose inhibit free oligosaccharide transport, di-N-acetylchitobiose is without effect. It is also shown that if the transport assay buffer is either depleted of calcium ions, or supplemented with the Ca2+/Mg2+ATPase inhibitor, thapsigargin, or with calcium ionophores, free oligosaccharide transport out of the endoplasmic reticulum is inhibited. These results demonstrate that the terminal nonreducing mannosyl residues of free polymannose-type oligosaccharides and not their N-acetylglucosamine-containing reducing termini, play an important role in the interaction of the free oligosaccharide with the transport machinery, and that this transport process requires the presence of calcium sequestered in the lumen of the endoplasmic reticulum.      

Development of Trypanosoma (M.) theileri in Tabanids

Thus far the life cycle of Trypanosoma (Megatrypanum) theileri has not been studied. We collected tabanids during the mass hatching, when only few tabanids are infected with trypanosomes. Tabanids were caught immediately after attacking a bait cow to serve as controls or after they had been allowed to engorge on the Trypanosoma (M.) theileri-infected cow. Tabanids were kept in the laboratory and used to study the developmental cycle of T. (M.) theileri in the tabanid gut. From day 1 to day 10 the presumably unfed controls and the engorged tabanids were dissected and cytological smears made from the mid- and hindgut. In total 2.6% (1/38) of the controls and 39% (23/59) of the engorged tabanids were positive for trypanosomes in the 1991 season. From day 1 to day 4 after engorgement trypanosomes were found in the midgut. Epimastigotes with a length of 29 μm on day 1 after infection multiplied by inequal division to form smaller epimastigotes of 26 μm on day 3. On day 4 morphologically indistinguishable trypanosomes of 21 μm total length were found in both mid- and hindgut. From day 5 to day 10 trypanosomes were found only in the hindgut in which the transformation to metacyclics was demonstrated, i.e. epimastigotes transformed to amastigote stages of 5 μm in total length.     

Relocation of the replication terminus, terC, of Bacillus subtilis to a new chromosomal site

The terC-deletion strain of Bacillus subtilis 168, SU153 [Iismaa and Wake, J. Mol. Biol. 195 (1987) 299-310] was used for the re-insertion of a 1.75-kb segment of DNA containing terC at a site approx. 25 kb from its original position. The relocated terC in the new strain, SU160, was oriented normally with respect to the approaching clockwise replication fork, and was positioned such that this fork was the first to reach it. The relocated terC was effective in causing arrest of the clockwise fork, as evidenced by the appearance of a unique DNA species with a characteristic mobility in agarose gel electrophoresis and with a predicted single-strand composition. Thus, the previously cloned 1.75-kb terC-containing segment [Smith et al., Gene 38 (1985) 9-17] has not been altered with respect to TerC function and contains sufficient sequence for this function. The findings reported here provide the opportunity for establishing the minimal and essential sequence features of terC, and for examining its possible polarity of action in causing fork arrest.