Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.     

The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli

The small subunit (SSU) of the ribosome of E. coli consists of a core of ribosomal RNA (rRNA) surrounded peripherally by ribosomal proteins (r-proteins). Ten of the 15 universally conserved SSU r-proteins possess nonglobular regions called extensions. The N-terminal noncanonically structured extension of S12 traverses from the solvent to intersubunit surface of the SSU and is followed by a more C-terminal globular region that is adjacent to the decoding center of the SSU. The role of the globular region in maintaining translational fidelity is well characterized, but a role for the S12 extension in SSU structure and function is unknown. We examined the effect of stepwise truncation of the extension of S12 in SSU assembly and function in vitro and in vivo. Examination of in vitro assembly in the presence of sequential N-terminal truncated variants of S12 reveals that N-terminal deletions of greater than nine amino acids exhibit decreased tRNA-binding activity and altered 16S rRNA architecture particularly in the platform of the SSU. While wild-type S12 expressed from a plasmid can rescue a genomic deletion of the essential gene for S12, rpsl; N-terminal deletions of S12 exhibit deleterious phenotypic consequences. Partial N-terminal deletions of S12 are slow growing and cold sensitive. Strains bearing these truncations as the sole copy of S12 have increased levels of free SSUs and immature 16S rRNA as compared with the wild-type S12. These differences are hallmarks of SSU biogenesis defects, indicating that the extension of S12 plays an important role in SSU assembly.     

Urea-induced dissociation and unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric and spectral studies.

Urea-induced dissociation and unfolding of manganese.glutamine synthetase (Mn.GS) have been studied at 37 degrees C (pH 7) by spectroscopic and calorimetric methods. In 0 to approximately 2 M urea, Mn.GS retains its dodecameric structure and full catalytic activity. Mn.GS is dissociated into subunits in 6 M urea, as evidenced by a 12-fold decrease in 90 degrees light scattering and a monomer molecular weight of 51,800 in sedimentation equilibrium studies. The light scattering decrease in 4 M urea parallels the time course of Trp exposure but occurs more rapidly than changes in secondary structure and Tyr exposure. Early and late kinetic steps appear to involve predominantly disruption of intra-ring and inter-ring subunit contacts, respectively, in the layered hexagonal structure of Mn.GS. The enthalpies for transferring Mn.GS into urea solutions have been measured by titration calorimetry. After correcting for the enthalpy of binding urea to the protein, the enthalpy of dissociation and unfolding of Mn.GS is 14 +/- 4 cal/g. A net proton uptake of approximately 50 H+/dodecamer accompanies unfolding reactions. The calorimetric data are consistent with urea binding to multiple, independent sites in Mn.GS and the number of binding sites increasing approximately 9-fold during the protein unfolding.     

Phylogeographic analysis of Pimoidae (Arachnida: Araneae) inferred from mitochondrial cytochrome c oxidase subunit I and nuclear 28S rRNA gene regions

Using mitochondrial DNA cytochrome c oxidase subunit I and nuclear DNA 28S rRNA data, we explored the phylogenetic relationships of the family Pimoidae (Arachnida: Araneae) and tested the North America to Asia dispersal hypothesis. Sequence data were analysed using maximum parsimony and Bayesian inference. A phylogenetic analysis suggested that vicariance, instead of dispersal, better explained the present distribution pattern of Pimoidae. Times of divergence events were estimated using penalized likelihood method. The dating analysis suggested that the emergence time of Pimoidae was approximately 140 million years ago (Ma). The divergence time of the North American and Asian species of Pimoa was approximately 110 Ma. Our phylogenetic hypothesis supports the current morphology‐based taxonomy and suggests that the cave dwelling might have played an important role in the speciation of pimoids in arid areas.     

Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding

We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ～10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.     

Homologs of eukaryotic Ras superfamily proteins in prokaryotes and their novel phylogenetic correlation with their eukaryotic analogs

Ras superfamily proteins are key regulators in a wide variety of cellular processes. Previously, they were considered to be specific to eukaryotes, and MglA, a group of obviously different prokaryotic proteins, were recognized as their only prokaryotic analogs or even ancestors. Here, taking advantage of quite a current accumulation of prokaryotic genomic databases, we have investigated the existence and taxonomic distribution of Ras superfamily protein homologs in a much wider prokaryotic range, and analyzed their phylogenetic correlation with their eukaryotic analogs. Thirteen unambiguous prokaryotic homologs, which possess the GDP/GTP-binding domain with all the five characteristic motifs of their eukaryotic analogs, were identified in 12 eubacteria and one archaebacterium, respectively. In some other archaebacteria, including four methanogenic archaebacteria and three Thermoplasmales, homologs were also found, but with the GDP/GTP-binding domains not containing all the five characteristic motifs. Many more MglA orthologs were identified than in previous studies mainly in delta-proteobacteria, and all were shown to have common unique features distinct from the Ras superfamily proteins. Our phylogenetic analysis indicated eukaryotic Rab, Ran, Ras, and Rho families have the closest phylogenetic correlation with the 13 unambiguous prokaryotic homologs, whereas the other three eukaryotic protein families (SRbeta, Sar1, and Arf) branch separately from them, but have a relatively close relationship with the methanogenic archaebacterial homologs and MglA. Although homologs were identified in a relative minority of prokaryotes with genomic databases, their presence in a relatively wide variety of lineages, their unique sequence characters distinct from those of eukaryotic analogs, and the topology of our phylogenetic tree altogether do not support their origin from eukaryotes as a result of lateral gene transfer. Therefore, we argue that Ras superfamily proteins might have already emerged at least in some prokaryotic lineages, and that the seven eukaryotic protein families of the Ras superfamily may have two independent prokaryotic origins, probably reflecting the 'fusion' evolutionary history of the eukaryotic cell.     

Evolved to be active: sulfate ions define substrate recognition sites of CK2alpha and emphasise its exceptional role within the CMGC family of eukaryotic protein kinases

CK2alpha is the catalytic subunit of protein kinase CK2 and a member of the CMGC family of eukaryotic protein kinases like the cyclin-dependent kinases, the MAP kinases and glycogen-synthase kinase 3. We present here a 1.6 A resolution crystal structure of a fully active C-terminal deletion mutant of human CK2alpha liganded by two sulfate ions, and we compare this structure systematically with representative structures of related CMGC kinases. The two sulfate anions occupy binding pockets at the activation segment and provide the structural basis of the acidic consensus sequence S/T-D/E-X-D/E that governs substrate recognition by CK2. The anion binding sites are conserved among those CMGC kinases. In most cases they are neutralized by phosphorylation of a neighbouring threonine or tyrosine side-chain, which triggers conformational changes for regulatory purposes. CK2alpha, however, lacks both phosphorylation sites at the activation segment and structural plasticity. Here the anion binding sites are functionally changed from regulation to substrate recognition. These findings underline the exceptional role of CK2alpha as a constitutively active enzyme within a family of strictly controlled protein kinases.     

α4β2 nicotinic acetylcholine receptors in the early postnatal mouse superior cervical ganglion

Heteropentameric nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. It is undisputed that α3 and β4 are the predominant subunits in the superior cervical ganglion (SCG); however, reports on the presence of receptors that contain α4 have been controversial. Here, we have searched for the presence of α4-containing nAChRs in the postnatal rat and mouse SCG. We now show by immunoprecipitation combined with radioligand binding that α4-containing receptors constitute about 20% of hetero-oligomeric nAChRs in postnatal Day 3 (P3) mice. However, already by P9, the level of α4 approaches zero. In contrast, the number of α4-containing receptors is close to zero in the rat SCG at all times investigated. Deletion of the β2 subunit by using α5β2-double knockout (KO) mice removes all α4-containing receptors, suggesting that in the postnatal mouse SCG, α4 co-assembles only with β2 but not with β4. α4β2 receptors are, on the other hand, up-regulated in the SCG of P3 α5β4-double KO mice, where they make up about 50% of receptors that bind [(3) H]-epibatidine. Nonetheless, receptors on the surface of SCG neurons from α5β4-double KO mice maintained for one to two days in culture comprise <10% of α4β2 and >90% of α3β2, as determined by patch clamp recordings with α4β2- and α3β2-specific ligands. We propose that in the P3 SCG of wild type mice, α3β4 (±α5) represent about 62% of receptors, whereas 17% are α3β2β4, and 21% are α4β2 (±α5) receptors.     

Molecular identification of schistosome intermediate hosts: case studies of Bulinus forskalii group species (Gastropoda: Planorbidae) from Central and East Africa

African freshwater snails of the genus Bulinus act as intermediate hosts for schistosomes, trematode parasites responsible for medical and veterinary forms of schistosomiasis. The relationship between these snails and their parasites is an intricate one, with particular species of snail susceptible to infection only by certain species of schistosome. In common with other self-fertile hermaphrodite gastropods, Bulinus consists of a number of closely related species complexes with restricted gene flow between populations of each taxon. Consequently, despite their medical and veterinary importance as intermediate hosts, unambiguous identification and differentiation of planorbid snails such as these remains problematic, often confounding attempts to define the distribution and evolutionary relationships of conchologically similar taxa. Here we consider how morphological methods of discrimination can be used in conjunction with molecular based approaches to improve snail identification, thereby achieving a better understanding of the epidemiology of schistosomiasis. Data are presented from Central and East African taxa which illustrate how PCR-based methods have begun to be used in combination with traditional analyses in an integrated approach to characterize the genus Bulinus , specifically the B. forskalii species group. Particular emphasis is given to the analyses of Random Amplified Polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI).     

Identification of SNPs and development of functional markers for LMW-GS genes at <Emphasis Type="Italic">Glu-D3</Emphasis> and <Emphasis Type="Italic">Glu-B3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GS) have great effect on wheat processing quality, but were numerous and difficult to dissect by SDS-PAGE. The development of functional markers may be the most effective way for a clear discrimination of different LMW-GS genes. In the present study, three different approaches were used to identify SNPs of different genes at Glu-D3 and Glu-B3 loci in bread wheat for the development of six STS markers (3 for Glu-D3 and 3 for Glu-B3 genes) that were validated with distinguished wheat cultivars. Firstly, seven LMW-GS gene sequences ( AY585350, AY585354, AY585355, AY585356, AY585349, AY585351 and AY585353 ) from Aegilops tauschii, the diploid donor of the D-genome of bread wheat, were chosen to design seven pairs of AS-PCR primers for Glu-D3 genes. By amplifying the corresponding genes from five bread wheat cultivars with different Glu-D3 alleles (a, b, c, d and e) and Ae. tauschii, a primer set, S13F2/S13R1, specific to the gene AY585356, was found to be positive to cultivars with alleles Glu-D3c and d. Nevertheless, the other five pairs of primers designed from AY585350, AY585349, AY585353, AY585354 and AY585355, respectively, did not produce specific PCR products to the cultivars tested. Secondly, all the PCR products from the five primer sets without specific characteristics were sequenced and an SNP from the gene AY585350 was detected in the cultivar Hartog, which resulted in the second STS marker S1F1/S1R3 specific to the allelic variant of AY585350. Thirdly, three Glu-D3 sequences (AB062851, AB062865 and AB062872) and three Glu-B3 sequences (AB062852, AB062853 and AB062860) defined by Ikeda et al. (2002) were chosen to query wheat EST and NR databases, and DNA markers were developed based on the putative SNPs among the sequences. Using this approach, four STS markers were developed and validated with 16-19 bread wheat cultivars. The primer set T1F4/T1R1 was also a Glu-D3 gene-specific marker for AB062872, while T2F2/T2R2, T5F3/T5R1 and T13F4/T13R3 were all Glu-B3 gene specific markers for AB062852, BF293671 and AY831800, respectively. The chromosomal locations of the six markers were verified by amplifying the genomic DNA of Ae. tauschii (DD), T. monococcum (AA) and T. turgidum (AABB) entries, as well as Chinese Spring and its group 1 chromosome nulli-tetrasomic lines. The results are useful to discriminate the corresponding Glu-D3 and Glu-B3 genes in wheat breeding programs.