Anticodon loop of tRNAPhe: structure, dynamics, and Mg2+ binding

The structure, dynamics, and Mg2+ binding reactions of the isolated anticodon hairpin loop from tRNAPhe (yeast) have been analyzed by fluorescence-detected temperature-jump relaxation, melting experiments, and equilibrium sedimentation. Most of the measurements were performed at an ionic strength of 0.15 M and at temperatures below 25 degrees C, where the hairpin loop proved to be stable. A relaxation effect with a time constant of approximately 100 microseconds, indicated by the Wye base fluorescence, is attributed to a conformational change of the anticodon loop and is very similar to a corresponding transition observed previously for the whole tRNAPhe molecule. A Mg2+ binding site reflected by an inner-sphere relaxation process and associated with a strong increase of the Wye base fluorescence closely resembles a corresponding site observed in the complete tRNAPhe and is attributed to a site in the anticodon loop identified by X-ray analysis. In addition to the Mg2+ site in the loop, which is associated with a binding constant of 2 X 10(3) M-1, the existence of sites with a higher affinity is demonstrated by an unusual relaxation effect, showing a minimum in the reciprocal time constant with increasing Mg2+ concentration. The experimental data can be described by a transition between two states and Mg2+ binding to both states resulting in a reaction cycle, which is extended by an additional Mg2+ binding reaction to one of the states. The unusual effect has not been observed for the complete tRNAPhe and is also not observed when Ca2+ is added instead of Mg2+. This result indicates the existence of a conformational change involving Mg2+ inner-sphere complexation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions

We describe a general method for the analysis of ligand-macromolecule binding equilibria for cases in which the interaction is monitored by a change in a signal originating from the ligand. This method allows the absolute determination of the average degree of ligand binding per macromolecule without any assumptions concerning the number of modes or states for ligand binding or the relationship between the fractional signal change and the fraction of bound ligand. Although this method is generally applicable to any type of signal, we discuss the details of the method as it applies to the analysis of binding data monitored by a change in fluorescence of a ligand upon binding to a nucleic acid. We apply the analysis to the equilibrium binding of Escherichia coli single-strand binding (SSB) protein to single-stranded nucleic acids, which is monitored by the quenching of the intrinsic tryptophan fluorescence of the SSB protein. With this method, one can quantitatively determine the relationship between the fractional signal change of the ligand and the fraction of bound ligand, LB/LT, and rigorously test whether the signal change is directly proportional to LB/LT. For E. coli SSB protein binding to single-stranded nucleic acids in its (SSB)65 binding mode [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Chrysogelos, S., & Griffith, J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5803], we show that the fractional quenching of the SSB fluorescence is equal to the fraction of bound SSB.     

Selective binding of amino acid residues to tRNAPhe

The interaction of amino acid amides with tRNAPhe was studied by measurements of the Wye base fluorescence. Binding of phenylalanine-, tyrosine- and tryptophan-amides leads to considerable quenching, whereas the amides of e.g. glycine and leucine do not induce quenching under the same conditions. Binding constants at 0.13 M salt - 100 M-1 for Phe-, 110 M-1 for Tyr- and 300 M-1 for Trp-amide - are about a factor of 6 higher than those evaluated from independent measurements for binding to simple single-stranded polynucleotides; the corresponding factor is 10 for double-stranded polynucleotides. Since the apparent enthalpy changes derived from measurements at different temperatures remains relatively low (-9 to -20 kJ/mol), the increased affinity appears to be mainly due to an increase of the entropy changes. Titration experiments performed in the presence of Mg2+ indicate cooperative interactions of the aromatic residues with the anticodon loop that are consistent with preferential binding to one of two loop conformations. Measurements of binding constants at different pH-values indicate the protonation of a tRNA residue in the tryptophanamide-tRNAPhe complex characterised by a pK value of about 7.0.     

RepSeq – A database of amino acid repeats present in lower eukaryotic pathogens

Background  

Amino acid repeat-containing proteins have a broad range of functions and their identification is of relevance to many experimental biologists. In human-infective protozoan parasites (such as the Kinetoplastid and Plasmodium species), they are implicated in immune evasion and have been shown to influence virulence and pathogenicity. RepSeq is a new database of amino acid repeat-containing proteins found in lower eukaryotic pathogens. The RepSeq database is accessed via a web-based application which also provides links to related online tools and databases for further analyses.     

Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands

Background  

G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro systems. Caenorhabditis elegans is a soil-dwelling, bacteria-feeding nematode that uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds, making this an ideal system for studying in vivo GPCR-ligand interactions. We sought to test this by functionally expressing two medically important mammalian GPCRs, somatostatin receptor 2 (Sstr2) and chemokine receptor 5 (CCR5) in the gustatory neurons of C. elegans.     

Biological Control of Sheep Parasites using Duddingtonia flagrans: Trials on Commercial Farms in Sweden

   

Trials were conducted on 3 commercial sheep farms in Sweden to assess the effect of administering spores of the nematode trapping fungus, Duddingtonia flagrans, together with supplementary feed to lactating ewes for the first 6 weeks from turn-out on pastures in spring. Also control groups of ewes, receiving only feed supplement, were established on all 3 farms. Groups were monitored by intensive parasitological investigation. The ewes and their lambs were moved in late June to saved pastures for summer grazing, the lambs receiving an anthelmintic treatment at this time. After approximately 6 weeks on summer pasture the lambs were weaned, treated a second time with anthelmintic, and returned to their original lambing pastures for finishing. Decisions as to when lambs were to be marketed were entirely at the discretion of the farmer co-operators. No difference in lamb performance was found between the two treatments on all three farms. This was attributed to the high levels of nutrition initially of the ewes limiting their post-partum rise in nematode faecal egg counts in spring, which in turn resulted in low levels of nematode infection on pastures throughout the autumn period. Additionally, pastures were of good quality for the lambs during the finishing period, so they grew at optimal rates as far as the farmers were concerned.     

Binding of six nucleotide cofactors to the hexameric helicase RepA protein of plasmid RSF1010. 2. Base specificity, nucleotide structure, magnesium, and salt effect on the cooperative binding of the cofactors

Interactions of the RepA hexameric helicase with nucleotide cofactors have been examined using nucleotide analogues, TNP-ADP and TNP-ATP, and unmodified nucleotides. Thermodynamic parameters for the interactions of modified and unmodified nucleotides have been obtained using quantitative fluorescence titration and competition titration methods. The intrinsic binding constant of ATP is by a factor of approximately 10 and approximately 1000 higher than the value observed for ADP and PO(4)(-). The data suggest that helicase acquires free-energy transducing capabilities when associated with the ssDNA, thus, forming a "holoenzyme". ATP binding is characterized by significantly stronger negative cooperativity than ADP. The cooperative interactions are predominantly induced through the specific interactions of the gamma phosphate and the ribose with the protein. The salt effect on cofactor binding indicates a very different nature of the intrinsic and cooperative interactions. Surprisingly, binding of Mg(2+), to both the cofactor and helicase, predominantly controls the ADP-RepA interactions. Mg(2+) cations seem to play a role in affecting the distribution of high and low ssDNA-affinity states, through the strong effect on the diphosphate versus triphosphate binding. The data indicate that Mg(2+) has a dual function in nucleotide-helicase interactions. At low [Mg(2+)], NTP binds stronger than NDP and the enzyme is predominantly in the high ssDNA-affinity state. At higher [Mg(2+)], NTP binds weaker than NDP and the helicase subunits can exist in alternating low- and high-affinity states that facilitate the efficient dsDNA unwinding. The RepA helicase shows a preference toward purine nucleotides. The cooperative interactions are independent of the type of the base.     

Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein

Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the gamma-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-gamma-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are approximately 2 and approximately 4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADP are approximately 2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.