Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage

The carboxy-terminal subdomains of the homodimeric EcoRV restriction endonuclease each bear a net charge of +4 and are positioned on the inner concave surface of the 50 degree DNA bend that is induced by the enzyme. A complete kinetic and structural analysis of a truncated EcoRV mutant lacking these domains was performed to assess the importance of this diffuse charge in facilitating DNA binding, bending, and cleavage. At the level of formation of an enzyme-DNA complex, the association rate for the dimeric mutant enzyme was sharply decreased by 10(3)-fold, while the equilibrium dissociation constant was weakened by nearly 10(6)-fold compared with that of wild-type EcoRV. Thus, the C-terminal subdomains strongly stabilize the enzyme-DNA ground-state complex in which the DNA is known to be bent. Further, the extent of DNA bending as observed by fluorescence resonance energy transfer was also significantly decreased. The crystal structure of the truncated enzyme bound to DNA and calcium ions at 2.4 A resolution reveals that the global fold is preserved and suggests that a divalent metal ion crucial to catalysis is destabilized in the active site. This may explain the 100-fold decrease in the rate of metal-dependent phosphoryl transfer observed for the mutant. These results show that diffuse positive charge associated with the C-terminal subdomains of EcoRV plays a key role in DNA association, bending, and cleavage.     

Critical comments on the one recent DNA bending model and its fit to electrophoretic data

The approach of Calladine, Drew and McCall (J. Mol. Biol 201, 127-137, 1988) for the investigation of DNA curvature has been analyzed. The authors relate their bending parameters to the experimental data on the basis of a new method of predicting electrophoretic anomalies from the trajectory of helical axis. This method of treating the DNA superhelix as a rigid body seems to be hardly applicable to the cases when superhelical turn is longer than or comparable to persistence length of straight DNA (approximately 150 bp). The extrapolation of experimental curves to plateau, done in a number of cases, seems somewhat arbitrary. Besides, alternative angle sets (e.g., with the roll angle on the AA step not equal to zero) having the same or even better fit to experimental data can be found even within the framework of the proposed approach. Another serious drawback of the model is its inability to account for new data (H.-S. Koo and D.M. Crothers. PNAS 85, 1763-1767, 1988), the predicted values of relative anomaly on (N8A6N5A6N4A6N7) multimers amounting to thousands.     

Chromatographic and electrophoretic analyses of papaya proteinases

Although it is known that unripe fruit from Carica papaya contains several proteinase enzymes which are used industrially, only one of these, papain, has been extensively characterized. Recently, the separate use of other enzymes of the family has been considered but information on their hydrodynamic properties is contradictory. The use of newer methods of separation has enabled us to separate a proteinase which runs slowly on acidic polyacrylamide gels (papain) from the four other proteinases. The proteinase which runs fastest on acidic polyacrylamide gels has an M, of 25 k and a pI of 11.0. This latter pI is the same as that of a proteinase which has an M, of 28 k and runs more quickly than papain but more slowly than chymopapain on acidic gels. We therefore have data showing that the proteinase enzymes from papaya can be classified by pI and M, into papain (pI 8.75, M, 23 k), chymopapains A and B (pI 10.3–10.4 or 10.6–10.7 and M, 24 k), papaya proteinase A (Ω) (pI 11.0, M, 24 k) and papaya proteinase β (pI 11.0, M, 28 K).     

DNA bending by the bulge defect

Comparative gel electrophoresis measurements were used to characterize DNA bending in molecules containing an extra adenosine on one strand, the so-called bulge defect. We used oligomers containing A6 tracts separated from the bulged base by varying numbers of nucleotides to determine the direction and magnitude of the bulge bend. Helix unwinding by the bulge was determined from the electrophoretic anomaly as a function of the size of the repeated monomers. We conclude that the bulge bend is 21 degrees +/- 3 degrees, primarily in the direction of tilt away from the bulged base. The total helical advance of the DNA at the bulge site is smaller than would be the case if the complementary T were present, corresponding to an unwinding by 25 degrees +/- 6 degrees. These values are in good agreement with the results of NMR and energy minimization studies of the bulged base in double-helical deoxyoligonucleotides [Woodson, S. A., & Crothers, D.M. (1988) Biochemistry 27, 3130-3141]     

DNA bending by bHLH charge variants

We wish to understand the role of electrostatics in DNA stiffness and bending. The DNA charge collapse model suggests that mutual electrostatic repulsions between neighboring phosphates significantly contribute to DNA stiffness. According to this model, placement of fixed charges near the negatively charged DNA surface should induce bending through asymmetric reduction or enhancement of these inter-phosphate repulsive forces. We have reported previously that charged variants of the elongated basic-leucine zipper (bZIP) domain of Gcn4p bend DNA in a manner consistent with this charge collapse model. To extend this result to a more globular protein, we present an investigation of the dimeric basic-helix–loop–helix (bHLH) domain of Pho4p. The 62 amino acid bHLH domain has been modified to position charged amino acid residues near one face of the DNA double helix. As observed for bZIP charge variants, DNA bending toward appended cations (away from the protein:DNA interface) is observed. However, unlike bZIP proteins, DNA is not bent away from bHLH anionic charges. This finding can be explained by the structure of the more globular bHLH domain which, in contrast to bZIP proteins, makes extensive DNA contacts along the binding face.     

Coupling of ligand binding and dimerization of helix-loop-helix peptides: spectroscopic and sedimentation analyses of calbindin D9k EF-hands

Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range lgK = 4.6-5.3.     

Shaping and bending of the avian neuroepithelium: morphometric analyses

Changes in the size and shape of the neuroepithelium were measured from serial transverse sections of 30 plastic-embedded chick embryos at stages 4-11. The neural plate folds into a neural tube during this period. Changes in volume, length, apical and basal widths, apical and basal surface areas, and thickness of the neuroepithelium were measured and correlated with the amount of folding that had occurred. These measurements were made to provide data for comparison with those available from other systems, to gain insight into the mechanisms of shaping and bending of the neuroepithelium, and to obtain normal parameters for eventual comparison with those obtained from embryos with induced neural tube defects. During stages 4-11, the volume, length, apical and basal surface areas, and lateral thickness of the neuroepithelium increase, whereas apical and basal widths and median thickness of the neuroepithelium decrease. Models are presented to demonstrate the effects of possible changes in neuroepithelial cell number, position, and size on the shaping of the neural plate.     

Capillary electrophoretic separation of proteins and peptides by ion-pairing with heptanesulfonic acid

Heptanesulfonic acid as ion-pairing agent was used for the separation of mixtures of low and high molecular mass peptides/proteins by capillary electrophoresis. The separation conditions used were: capillary 37 cm (30 cm to the detector) x 75 microm i.d., voltage 10 kV, phosphate buffer 50 mmol/l, ion-pairing agent heptanesulfonic acid at three different concentrations, namely, 0, 20 or 100 mmol/l, pH 2.5. The separation reflected the ion-pairing equilibria between the ion-pairing agent and the peptide/protein analytes. The influence of ion-pairing on sample mobility (running time) was more pronounced in case of the higher-molecular peptides as compared to the low molecular ones. This difference offers the possibility to separate low and high molecular peptides/proteins that under the absence of the ion-pairing agent would co-migrate. The principle of this approach was demonstrated on a randomly selected set of peptides/proteins; the practical applicability was demonstrated on a set of CNBr peptides arising from a naturally occurring mixture of collagen types I and III.     

Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector

The binding of a protein to its specific sequence, borne on a DNA fragment, retards the mobility of the fragment in a characteristic way during gel electrophoresis. If the protein induces bending in the DNA, the contortion can also be monitored by gel electrophoresis, because the amount of retardation of the mobility of the DNA-protein complex is dependent upon the position and the degree of the bend induced in the DNA fragment [Wu and Crothers, Nature 308 (1984) 509-513]. We have constructed a plasmid, pBend2, which can generate a large number of DNA fragments of identical length in which the protein-binding nucleotide sequence is located in circular permutations. The vector contains two identical DNA segments containing 17 restriction sites in a direct repeat spanning a central region containing cloning sites. The protein-binding sequence is inserted at one of these cloning sites. To investigate the functional significance of bending, we have compared, using pBend2, the cAMP.cAMP-receptor protein (CPR)-induced bending of CRP-binding sites found in five different genes of Escherichia coli. We have also shown that the bacteriophage lambda 0R1 operator DNA is bent when complexed with the CI or Cro repressor of the phage.     

The DNA bending by acetylaminofluorene residues and by apurinic sites

We have studied the distortions induced in double-stranded oligonucleotides by covalently bound acetylaminofluorene residues and by apurinic sites. Within the acetylaminofluorene-modified oligonucleotide three base-pairs are unpaired as detected by the chemical probes chloroacetaldehyde and osmium tetroxide. These two probes reveal that the bases adjacent to the apurinic site are paired. In both the modified double-stranded oligonucleotides, the backbone on the 5' side of the modification is more reactive with 1,10-phenanthroline copper than the backbone on the 3' side. On polyacrylamide gels, the ligated multimers of acetylaminofluorene or apurinic site-modified oligonucleotides migrate slower than the multimers of the unmodified oligonucleotides. It is suggested that the acetylaminofluorene-modified guanine residues and the apurinic sites behave more as hinge joints than as the centres of directed bends.     

DNA binding and bending by HMG boxes: energetic determinants of specificity

To clarify the physical basis of DNA binding specificity, the thermodynamic properties and DNA binding and bending abilities of the DNA binding domains (DBDs) of sequence-specific (SS) and non-sequence-specific (NSS) HMG box proteins were studied with various DNA recognition sequences using micro-calorimetric and optical methods. Temperature-induced unfolding of the free DBDs showed that their structure does not represent a single cooperative unit but is subdivided into two (in the case of NSS DBDs) or three (in the case of SS DBDs) sub-domains, which differ in stability. Both types of HMG box, most particularly SS, are partially unfolded even at room temperature but association with DNA results in stabilization and cooperation of all the sub-domains. Binding and bending measurements using fluorescence spectroscopy over a range of ionic strengths, combined with calorimetric data, allowed separation of the electrostatic and non-electrostatic components of the Gibbs energies of DNA binding, yielding their enthalpic and entropic terms and an estimate of their contributions to DNA binding and bending. In all cases electrostatic interactions dominate non-electrostatic in the association of a DBD with DNA. The main difference between SS and NSS complexes is that SS are formed with an enthalpy close to zero and a negative heat capacity effect, while NSS are formed with a very positive enthalpy and a positive heat capacity effect. This indicates that formation of SS HMG box-DNA complexes is specified by extensive van der Waals contacts between apolar groups, i.e. a more tightly packed interface forms than in NSS complexes. The other principal difference is that DNA bending by the NSS DBDs is driven almost entirely by the electrostatic component of the binding energy, while DNA bending by SS DBDs is driven mainly by the non-electrostatic component. The basic extensions of both categories of HMG box play a similar role in DNA binding and bending, making solely electrostatic interactions with the DNA.     

DNA bending induced by DNA (cytosine-5) methyltransferases

DNA bending induced by six DNA (cytosine-5) methyltransferases was studied using circular permutation gel mobility shift assay. The following bend angles were obtained: M.BspRI (GG^m5CC), 46–50°; M.HaeIII (GG^m5CC), 40–43°; M.SinI (GGW^m5CC), 34–37°; M.Sau96I (GGN^m5CC), 52–57°; M.HpaII (C^m5CGG), 30°; and M.HhaI (G^m5CGC), 13°. M.HaeIII was also tested with fragments carrying a methylated binding site, and it was found to induce a 32° bend. A phase-sensitive gel mobility shift assay, using a set of DNA fragments with a sequence-directed bend and a single methyltransferase binding site, indicated that M.HaeIII and M.BspRI bend DNA toward the minor groove. The DNA curvature induced by M.HaeIII contrasts with the lack of DNA bend observed for a covalent M.HaeIII–DNA complex in an earlier X-ray study. Our results and data from other laboratories show a correlation between the bending properties and the recognition specificities of (cytosine-5) methyltransferases: enzymes recognizing a cytosine 3′ to the target cytosine tend to induce greater bends than enzymes with guanine in this position. We suggest that the observed differences indicate different mechanisms employed by (cytosine-5) methyltransferases to stabilize the helix after the target base has flipped out.     

Specificity of Kirkman-Robbins hepatoma non-histone chromatin proteins: electrophoretic and immunological analyses

The specificity of Kirkman-Robbins hepatoma and hamster liver non-histone chromatin proteins has been studied by comparing polypeptide patterns in polyacrylamide gel electrophoresis and by their immunological activity in the complement fixation test. Non-histone proteins were separated from DNA with a polyethylene glycol-dextran mixture and fractionated by hydroxylapatite chromatography into three classes named NHCP1, NHCP2, and NHCP3. Electrophoretic analysis indicated that among the non-histone proteins of Kirkman-Robbins hepatoma and hamster liver differences mainly of a quantitative nature can be observed. However, the polypeptides with molecular weight 25 000, 31 000, 36 000, 73 000 in NHCP1; 20 000, 40 000 in NHCP2 and 20 000, 23 000, 32 000, 38 000, 44 000, 75 000, 80 000 in NHCP3 were found to be specific for hepatoma chromatin. Application of antibodies against NHCP1, NHCP2 and dehistonized chromatin of Kirkman-Robbins hepatoma revealed that the highest specificity of NHCP2 eluted from hydroxylapatite with 100 mM phosphate buffer at pH 6.8. The NHCP1 of hepatoma shares some common antigenic determinants with analogous proteins of liver. On the other hand non-histone proteins specific for hepatoma dehistonized chromatin can be localized in the NHCP3 and partially in the NHCP1 fractions.     

Variation of the intercalating proline in artificial peptides mimicking the DNA binding and bending IHF protein

The integration host factor (IHF) is a protein which sequence specifically induces a bend of double-stranded DNA by more than 160°. Based on IHF as lead structure, a peptide mimic was introduced resembling the positively charged body of the protein by a lysine dendrimer and the minor groove recognition loop by a cyclopeptide. The proline located close to the tip of the recognition loop intercalates between the base pair plane. It was modified in order to evaluate the influence of the side chain residue with respect to size (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), aromaticity (phenylalanine), conformation of the five-membered ring [(4R)-fluoroproline, (4S)-fluoroproline, 3,4-dehydroproline], and the peptide backbone conformation (α-methylproline) on binding dsDNA and bending the double strand. Binding and bending studies were carried out by fluorescence resonance energy transfer experiments and gel electrophoresis using DNA sequences prepared by PCR with the IHF binding site in central or terminal position. Whereas aromatic residues and α-methylproline were not tolerated as proline substitute, incorporation of (4S)-fluoroproline and 3,4-dehydroproline provided enhanced binding.     

Infra-red spectroscopic analyses of banana waste degraded by oyster mushroom

Carbon, hydrogen and nitrogen analyses of banana leaf and pseudostem biomass revealed their potentiality as substrates for microorganisms. Infra-red (IR) spectra of both biomass show presence of cellulose, xylan and lignin. IR spectra of leaf and pseudostem biomass degraded in solid state fermentation (SSF) by two Pleurotus species (P. sajor-caju and P. ostreatus) for 40 days showed the utilization of cellulose, xylan and lignin by these microbes. Dynamics of various lignocellulolytic enzymes of Pleurotus species and analyses of carbon, hydrogen and nitrogen contents of degraded biomass supported the same. Both the Pleurotus species exhibited lignin consumption ability on both the substrates.     

Characterization of copper atoms in bilirubin oxidase by spectroscopic analyses

Bilirubin oxidase [EC 1.3.3.5], purified from the culture medium of Myrothecium verrucaria, was found to contain two blue copper atoms per protein molecule with a molecular weight of ca. 52 kDa. The two copper atoms were estimated to be in the all cupric state by the cuproine colorimetric method and also atomic absorption analysis. We could remove the reduce cuprous ions from the holo enzyme by adding ascorbate, followed by a KCN solution, yielding an apo-enzyme with no activity. The apo-enzyme can be reconstituted with Cu or other divalent cations such as Co, Fe, and Cd, with accompanying recovery of the enzyme activity. The activity recovery depended upon the species of cation employed; Cu being most effective, an almost 100% recovery, and Cd the least, only a 25% recovery. We could obtain information on the copper ions and their coordination structure by spectroscopic analyses of the apo- and reconstituted enzymes, obtaining such as absorption, CD, MCD, and XPS spectra. The bilirubin oxidase catalyzed-reaction was a second order reaction with respect to copper bound with protein. The donor set was of the CuSS*N2 (S = Cys, S* = Met, N = His) type, i.e., the same as in the case of blue copper proteins. On studying the Co-substituted enzyme, it was revealed that the copper site of the enzyme had a 4-coordinated structure.