Natively unfolded nucleic acid binding P8 domain of SeMV polyprotein 2a affects the novel ATPase activity of the preceding P10 domain

Open reading frame (ORF) 2a of Sesbania mosaic virus (SeMV) codes for polyprotein 2a (Membrane anchor-protease-VPg-P10-P8). The C-terminal domain of SeMV polyprotein 2a was cloned, expressed and purified in order to functionally characterize it. The protein of size 8 kDa (P8) domain, like viral protein genome linked (VPg), was found to be natively unfolded and could bind to nucleic acids.Interestingly, P10-P8 but not P8 showed a novel Mg2+ dependent ATPase activity that was inhibited in the presence of poly A. In the absence of P8, the ATPase activity of the protein of size 10 kDa (P10) domain was reduced suggesting that the natively unfolded P8 domain influenced the P10 ATPase.

Immunospecific binding of tRNA precursors containing N6-(delta 2-isopentenyl) adenosine

Antibodies specific for N6-(delta 2-isopentenyl) adenosine (i6A) were immobilized on Sepharose and this adsorbent (Sepharose-anti-i6A) was used to selectively isolate bacteriophage T4 tRNA precursors containing i6A/ms2i6A from an unfractionated population of 32P-labeled T4 RNAs. The results showed that antibodies to i6A selectively bound only those tRNA precursors containing i6A/ms2i6A. Binding of tRNA precursors by antibody and specificity of the binding was assessed by membrane binding using 32P-labeled tRNA precursor. Binding was highly specific for i6A/ms2i6A residues in the tRNA precursors. This binding can be used to separate modified from unmodified precursor RNAs and to study the biosynthetic pathways of tRNA precursors.

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

13C nuclear magnetic resonance studies of the binding of alkali and alkaline earth metal salts to amides

3C resonances of carbonyl and methyl groups in amides are shifted down-field on interaction with alkali and alkaline earth metal salts. The magnitude of the shift depends on the ionic potential of the cation. Ions like Li+ bind to the amide carbonyl group both in neat amide solutions as well as in concentrated salt solutions in water.

Fluorescence polarization as a tool to study lectin-sugar interaction. An investigation of the binding of 4-methylumbelliferyl beta-D-galactopyranoside to Abrus precatorious agglutinin

Polarization of ligand fluorescence was used to study the binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp) to Abrus precatorious agglutinin. The binding of the fluorescent sugar to the lectin led to considerable polarization of the MeUmb-Galp fluorescence, which was also quenched by about 30% on binding to the lectin. The binding of the fluorescent sugar was carbohydrate-specific, as evidenced by inhibition of both fluorescence polarization and quenching when lectin was preincubated with lactose. The association constant as determined by fluorescence polarization is 1.42 x 10(4) M-1 at 25 degrees C and is in excellent agreement with those determined by fluorescence quenching (Ka = 1.51 x 10(4) M-1) and equilibrium dialysis (Ka = 1.62 x 10(4) M-1) at 25 degrees C. The numbers of binding sites as determined by fluorescence polarization, quenching and equilibrium dialysis agree very well with one another, n being equal to 2.0 +/- 0.05. The consistency between the association constant value determined by fluorescence polarization, quenching and equilibrium dialysis shows the validity of this approach to study lectin-sugar interaction.

Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing

We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.

Binding and regulatory properties of phosphofructokinase from swine kidney

The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P2 reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P2 the reaction showed a sigmoidal dependence on fructose-6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate. The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The KD for fructose 6-P was 13 microM and in the presence of 0.5 mM ATP it increased to 27 microM. The addition of 0.3 mM citrate also increased the KD for fructose 6-P to about 40 microM. AMP, 10 microM, decreased the KD to 5 microM and the addition of fructose 2,6-phosphate decreased the KD for fructose 6-P to 0.9 microM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The KD of the site with the highest affinity for ATP was 4 microM, and it increased to 15 microM in the presence of fructose 2,6-bisphosphate. The addition of 50 microM fructose 1,6-bisphosphate increased the KD for ATP to 5.9 microM. AMP increased the KD to 5.9 microM whereas 0.3 mM citrate decreased the KD for ATP to about 2 microM.(ABSTRACT TRUNCATED AT 400 WORDS).

Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure

When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.

The brain serotonin transporter binding in young adults; methodological considerations and association with Bulimia Nervosa and acquired obesity

The neurotransmitter serotonin (5-HT) modulates many functions important for life, e.g., appetite and body temperature, and controls development of the neural system. Disturbed 5-HT function has been implicated in mood, anxiety and eating disorders. The serotonin transporter (SERT) controls the amount of effective 5-HT by removing it from the extracellular space. Radionuclide imaging methods single photon emission tomography (SPET) and positron emission tomography (PET) enable studies on the brain SERTs. This thesis concentrated on both methodological and clinical aspects of the brain SERT imaging using SPET. The first study compared the repeatability of automated and manual methods for definition of volumes of interest (VOIs) in SERT images. The second study investigated within-subject seasonal variation of SERT binding in healthy young adults in two brain regions, the midbrain and thalamus. The third study investigated the association of the midbrain and thalamic SERT binding with Bulimia Nervosa (BN) in female twins. The fourth study investigated the association of the midbrain and hypothalamic/thalamic SERT binding and body mass index (BMI) in monozygotic (MZ) twin pairs. Two radioligands for SERT imaging were used: [123I]ADAM (studies I-III) and [123I]nor-beta-CIT (study IV). Study subjects included young adult MZ and dizygotic (DZ) twins screened from the FinnTwin16 twin cohort (studies I-IV) and healthy young adult men recruited for study II. The first study validated the use of an automated brain template in the analyses of [123I]ADAM images and proved automated VOI definition more reproducible than manual VOI definition. The second study found no systematic within-subject variation in SERT binding between scans done in summer and winter in either of the investigated brain regions. The third study found similar SERT binding between BN women (including purging and non-purging probands), their unaffected female co-twins and other healthy women in both brain regions; in post hoc analyses, a subgroup of purging BN women had significantly higher SERT binding in the midbrain as compared to all healthy women. In the fourth study, MZ twin pairs were divided into twins with higher BMI and co-twins with lower BMI; twins with higher BMI were found to have higher SERT binding in the hypothalamus/thalamus than their leaner co-twins. Our results allow the following conclusions: 1) No systematic seasonal variation exists in the midbrain and thalamus between SERT binding in summer and winter. 2) In a population-based sample, BN does not associate with altered SERT status, but alterations are possible in purging BN women. 3) The higher SERT binding in MZ twins with higher BMIs as compared to their leaner co-twins suggests non-genetic association between acquired obesity and the brain 5-HT system, which may have implications on feeding behavior and satiety.

Binding of nucleobases with single-walled carbon nanotubes: Theory and experiment

We report the binding energy of various nucleobases (guanine (G), adenine (A), thymine (T) and cytosine (C)) with (5,5) single-walled carbon nanotube (SWNT) calculated using first-principle Hartre–Fock method (HF) together with classical force field. The binding energy without including the solvation effects of water decreases in the order G>A>T>C. The inclusion of solvation energy changes the order of binding preference to be G>T>A>C. Using isothermal titration (micro) calorimetry experiments, we also show the relative binding affinity to be T>A>C, in agreement with our calculations.

Design, Synthesis, and DNA Binding Properties of Photoisomerizable Azobenzene−Distamycin Conjugates: An Experimental and Computational Study

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene−distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electron-withdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT)10CG)]2 DNA were performed to rationalize the nature of binding of these conjugates.

Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to or 70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70).

Preterm delivery and selected biomarkers : phosphorylated insulin-like growth factor-binding protein-1 and matrix metalloproteinase-8 – in cervical fluid

Premature delivery is a major cause of neonatal morbidity and mortality. The incidence of premature deliveries has increased around the world. In Finland 5.3%, or about 3,000 children per year are born prematurely, before 37 weeks of gestation. The corresponding figure in the United States is about 13%. The morbidity and mortality are highest among infants delivered before 32 weeks of gestation - about 600 children each year in Finland. Approximately 70% of premature deliveries are unexplained. Preterm delivery can be caused by an asympto-matic infection between uterus and the fetal membranes, such can begin already in early pregnancy. It is difficult to predict preterm delivery, and many patients are therefore unnecessarily admitted to hospital for observation and exposed to medical treatments. On the other hand, the high risk women should be identified early for the best treatment of the mother and preterm infant. --- In the prospective study conducted at the Department of Obstetric and Gynecology, Helsinki University Central Hospital two biochemical inflammation related markers were measured in the lower genital tract fluids of asymp-tomatic women in early and mid pregnancy in an order to see whether these markers could identify women with an increased risk of preterm delivery. These biomarkers were phosphorylated insulin-like growth factor binding protein-1 (phIGFBP-1) and matrix metalloproteinase-8 (MMP-8). The study involved 5180 asymptomatic pregnant women, examined during the first and second ultrasound screening visits. The study samples were taken from the vagina and cervicix. In addition, 246 symptomatic women were studied (pregnancy weeks 22 – 34). The study showed that increased phIGFBP-1 concentration in cervical canal fluid in early pregnancy increased the risk for preterm delivery. The risk for very premature birth (before 32 weeks of gestation) was nearly four-fold. Low MMP-8 concentration in mid pregnancy increased the risk of subsequent premature preterm rupture of fetal membranes (PPROM). Significantly high MMP-8 concentrations in the cervical fluid increased the risk for prema-ture delivery initiated by preterm labour with intact membranes. Among women with preterm contractions the shortened cervical length measured by ultrasound and elevated cervical fluid phIGFBP-1 both predicted premature delivery. In summary, because of the relatively low sensitivity of cervical fluid phIGFBP-1 this biomarker is not suitable for routine screening, but provides an additional tool in assessing the risk of preterm delivery. Cervical fluid MMP-8 is not useful in early or mid pregnancy in predicting premature delivery because of its dual role. Further studies on the role of MMP-8 are therefore needed. Our study confirms that phIGFBP-1 testing is useful in predicting pre-term delivery.