Evolutionary Dynamics of Human Rotaviruses: Balancing Reassortment with Preferred Genome Constellations

Group A human rotaviruses (RVs) are a major cause of severe gastroenteritis in infants and young children. Yet, aside from the genes encoding serotype antigens (VP7; G-type and VP4; P-type), little is known about the genetic make-up of emerging and endemic human RV strains. To gain insight into the diversity and evolution of RVs circulating at a single location over a period of time, we sequenced the eleven-segmented, double-stranded RNA genomes of fifty-one G3P[8] strains collected from 1974 to 1991 at Children''s Hospital National Medical Center, Washington, D. C. During this period, G1P[8] strains typically dominated, comprising on average 56% of RV infections each year in hospitalized children. A notable exception was in the 1976 and 1991 winter seasons when the incidence of G1P[8] infections decreased dramatically, a trend that correlated with a significant increase in G3P[8] infections. Our sequence analysis indicates that the 1976 season was characterized by the presence of several genetically distinct, co-circulating clades of G3P[8] viruses, which contained minor but significant differences in their encoded proteins. These 1976 lineages did not readily exchange gene segments with each other, but instead remained stable over the course of the season. In contrast, the 1991 season contained a single major clade, whose genome constellation was similar to one of the 1976 clades. The 1991 clade may have gained a fitness advantage after reassorting with as of yet unidentified RV strain(s). This study reveals for the first time that genetically distinct RV clades of the same G/P-type can co-circulate and cause disease. The findings from this study also suggest that, although gene segment exchange occurs, most reassortant strains are replaced over time by lineages with preferred genome constellations. Elucidation of the selective pressures that favor maintenance of RVs with certain sets of genes may be necessary to anticipate future vaccine needs.     

Anaerobic growth of Paracoccus denitrificans requires cobalamin: characterization of cobK and cobJ genes

A pleiotropic mutant of Paracoccus denitrificans, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated. This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B(12)) or cobinamide to the media or by growth on rich media. Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to cobK of Pseudomonas denitrificans, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway. Convergently transcribed with cobK is a gene homologous to cobJ of Pseudomonas denitrificans, which encodes precorrin-3b methyltransferase. The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type P. denitrificans (which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions. Comparison of the cobK and cobJ genes with their orthologues suggests that P. denitrificans uses the aerobic pathway for cobalamin synthesis. It is paradoxical that under anaerobic growth conditions, P. denitrificans appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis. Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media. These observations provide evidence that P. denitrificans expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions.     

Site mutations disrupt inter-helical H-bonds (alpha14W-alpha67T and beta15W-beta72S) involved in kinetic steps in the hemoglobin R-->T transition without altering the free energies of oxygenation

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between alpha67T and alpha14W and between beta72S and beta15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 degrees C show that rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A) exhibit oxygen-binding properties similar to those of Hb A. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (alphaT67V), rHb (betaS72A), rHb (alphaT67V, betaS72A), and Hb A have similar quaternary structures in the alpha(1)beta(2) subunit interfaces. In particular, the inter-subunit H-bonds between alpha42Tyr and beta99Asp and between beta37Trp and alpha94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the alpha- and beta-chains in the mutants. A comparison of the exchangeable 1H resonances of Hb A with those of these three rHbs suggests that alpha67T and beta72S are H-bonded to alpha14W and beta15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at alphaT67V and betaS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.     

Vibrational and electronic couplings in ultraviolet resonance Raman spectra of cyclic peptides

Ultraviolet resonance Raman (UVRR) spectra are reported for a series of cyclic amides. 2-Azacyclotridecone, which has a 13-membered ring, shows a classic trans-amide UVRR spectral pattern, with comparable enhancement of the amide modes II, III and S. When the ring is diminished to eight (epsilon -caprolactam) or seven (2-azacyclooctanone) members, this pattern is replaced by a single strong band near 1497 cm(-1), characteristic of the Cz-N stretch of a cis-amide vibration (amide IIc). Further shrinkage of the ring decreases the amide IIc frequency. It is lowered over 100 cm(-1) to 1389 cm(-1) in the case of a 4-membered ring (2-azaidine), reflecting diminution of the Cz-N bond order due to ring strain and pyramidalization of the C and N atoms. At the same time the amide Ic (Cz=O stretching) frequency increases, reflecting the localization of the Cz=O double bond. Also the sensitivity to hydrogen-deuterium exchange reverses for amide Ic and IIc modes as ring size decreases. The UV absorption maximum, which is red-shifted for cis-relative to trans-amides, shifts increasingly to the blue as the ring size decreases, again reflecting localization of the pi bonding. In the case of amides with 5- and 6-membered rings (2-pyrrolidinone and delta-eloctam) multiple UVRR bands are seen in the amide IIc region, whose relative intensities are temperature-dependent. These are assigned to conformers in which different members of the ring are out of the mean plane, resulting in variable perturbations of the amide bond. The cyclic dipeptides cyclo(Gly-Gly) and cyclo(Gly-Pro) have perturbed amide IIc frequencies, reflecting the kinematic mixing of the amide coordinates into in- and out-of-phase modes. Excitation profiles reveal electronic mixing as well, with the transition dipoles adding for the in-phase and cancelling for the out-of-phase modes.     

FeNO structure in distal pocket mutants of myoglobin based on resonance Raman spectroscopy

FeNO vibrational frequencies were investigated for a series of myoglobin mutants using isotope-edited resonance Raman spectra of (15/14)NO adducts, which reveal the FeNO and NO stretching modes. The latter give rise to doublet bands, as a result of Fermi resonances with coincident porphyrin vibrations; these doublets were analyzed by curve-fitting to obtain the nuNO frequencies. Variations in nuNO among the mutants correlate with the reported nuCO variations for the CO adducts of the same mutants. The correlation has a slope near unity, indicating equal sensitivity of the NO and CO bonds to polar influences in the heme pocket. A few mutants deviate from the correlation, indicating that distal interactions differ for the NO and CO adducts, probably because of the differing distal residue geometries. In contrast to the strong and consistent nuFeC/nuCO correlation found for the CO adducts, nuFeN correlates only weakly with nuNO, and the slope of the correlation depends on which residue is being mutated. This variability is suggested to arise from steric interactions, which change the FeNO angle and therefore alter the Fe-NO and N-O bond orders. This effect is modeled with Density Functional Theory (DFT) and is rationalized on the basis of a valence isomer bonding model. The FeNO unit, which is naturally bent, is a more sensitive reporter of steric interactions than the FeCO unit, which is naturally linear. An important additional factor is the strength of the bond to the proximal ligand, which modulates the valence isomer equilibrium. The FeNO unit is bent more strongly in MbNO than in protein-free heme-NO complexes because of a combination of a strengthened proximal bond and distal interactions.     

Protein glycosylation: nature,distribution, enzymatic formation,and disease implications of glycopeptide bonds

Formation of the sugar-amino acid linkage is a crucial event in the biosynthesis of the carbohydrate units of glycoproteins. It sets into motion a complex series of posttranslational enzymatic steps that lead to the formation of a host of protein-bound oligosaccharides with diverse biological functions. These reactions occur throughout the entire phylogenetic spectrum, ranging from archaea and eubacteria to eukaryotes. It is the aim of this review to describe the glycopeptide linkages that have been found to date and specify their presence on well-characterized glycoproteins. A survey is also made of the enzymes involved in the formation of the various glycopeptide bonds as well as the site of their intracellular action and their affinity for particular peptide domains is evaluated. This examination indicates that 13 different monosaccharides and 8 amino acids are involved in glycoprotein linkages leading to a total of at least 41 bonds, if the anomeric configurations, the phosphoglycosyl linkages, as well as the GPI (glycophosphatidylinositol) phosphoethanolamine bridge are also considered. These bonds represent the products of N- and O-glycosylation, C-mannosylation, phosphoglycation, and glypiation. Currently at least 16 enzymes involved in their formation have been identified and in many cases cloned. Their intracellular site of action varies and includes the endoplasmic reticulum, Golgi apparatus, cytosol, and nucleus. With the exception of the Asn-linked carbohydrate and the GPI anchor, which are transferred to the polypeptide en bloc, the sugar-amino acid linkages are formed by the enzymatic transfer of an activated monosaccharide directly to the protein. This review also deals briefly with glycosidases, which are involved in physiologically important cleavages of glycopeptide bonds in higher organisms, and with a number of human disease states in which defects in enzymatic transfer of saccharides to protein have been implicated.