Biomedical subjects

J C Maurizot

Publications and source records attributed to J C Maurizot.

At least 19 recordsLinked to original sources

PubMed1996

Distamycin A complexation with a nucleic acid triple helix.

The interaction of the minor groove binding drug distamycin with the T-A-T triple helix and the A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by the oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3'-phosphate of one strand and the 5'-phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide to be in a duplex form with a dangling arm. Circular dichroism spectroscopy demonstrates that the distamycin A molecule can bind to the triple-stranded form of this oligonucleotide. Spectral analysis shows that the bound distamycin exhibits a conformation and an environment slightly different from those which are observed when the drug is bound to the corresponding double-stranded structure. Furthermore, a second type of complex which is observed in the double-strand binding (two stacked distamycins in the minor groove) is not observed with the triple-stranded host. When distamycin is added to the triplex made of unbridged chains (dA)12 + 2(dT)12, the triplex dissociates to give a double-stranded structure. Thermal denaturation experiments demonstrate that distamycin binding destabilizes the triplex whereas it stabilizes the duplex. These results are compared with those obtained by the same experimental approaches on other minor groove binding drugs.

Antiviral Agents↗

PubMed1996

Preferential binding of the archaebacterial histone-like MC1 protein to negatively supercoiled DNA minicircles.

The interaction of the archaebacterial MC1 protein with 207 bp negatively supercoiled DNA minicircles has been examined by gel retardation assays and compared to that observed with the relaxed DNA minicircle. MC1 binding induces a drastic DNA conformational change of each minicircle, leading to an increase of the electrophoretic mobility of the DNA. A slight increase in salt concentration enhances the amount of bound MC1, and high NaCl concentrations are required to dissociate the complexes. Furthermore, the salt effect on binding depends on the supercoiling state of the DNA. The dissociation rates decrease with increasing linking difference of the minicircles relative to their relaxed configuration to reach a maximum at -2 turns. In addition, differences between the topoisomers are also observed in terms of stoichiometry of the strongest complexes. So with the -2 topoisomer the complex with two MC1 molecules is the most stable, while with the -1 and -3 topoisomers, the strongest ones are those with one MC1 molecule per DNA ring.

Archaeal Proteins↗

PubMed1995

Conformational changes of DNA minicircles upon the binding of the archaebacterial histone-like protein MC1.

Binding of the archaebacterial histone-like protein MC1 to DNA minicircles has been examined by gel retardation and electron microscopy. MC1 preferentially binds to a 207-base pair relaxed DNA minicircle as compared with the linear fragment. Random binding is observed at very low ionic strength, and a slight increase in salt concentration highly favors the formation of a complex that corresponds to the binding of two MC1 molecules per DNA ring. Measurements of dissociation rates show that this complex is remarkably stable, and electron microscopy reveals that it is characterized by two diametrically opposed kinks. These results are discussed in regard to the mechanisms by which MC1 affects DNA structure.

Archaeal Proteins↗

PubMed1995

Interaction between cisplatin-modified DNA and the HMG boxes of HMG 1: DNase I footprinting and circular dichroism.

The interactions between the two boxes A and B of HMG 1 and cis-diamminedichloroplatinum(II)-modified DNA containing a single intrastrand cross-link at the d(GpG) site were studied by DNase I footprinting and circular dichroism. The DNAase I cleavage patterns of the HMG box-platinated DNA complexes are identical, the two boxes inhibiting the DNase I cutting over at least 15 and 12 nucleotide residues in the platinated strand and the complementary strand, respectively. As judged by circular dichroism, the two boxes have the same alpha-helical content (56%) and they induce the same conformational changes in the platinated DNA.

Amino Acid Sequence↗

PubMed1995

The secondary structure of the insect defensin A depends on its environment. A circular dichroism study.

Defensin A is an inducible antibacterial protein isolated from the larvae of Phormia terranovae. The conformation of defensin A has been previously determined by two-dimensional 1H-NMR for concentrations in the range of 4-8 mM in water (Bonmatin JM et al (1992) J Biomol NMR 2, 235-256). CD spectroscopic data of defensin A at lower concentrations (10(-5) to 10(-3) M) are reported herein. The ellipticity in the 200-240 nm wavelength range for various solvents varies as follows: acetonitrile < water < methanol < HFIP. The magnitude of theta 222 is strongly dependent on defensin concentration in a buffer solution, suggesting an aggregation process. The helical content of defensin A is maximum at a pH value range (7.5-8) for which the optimum antibacterial activity was observed (Cociancich S et al (1993) J Biol Chem 268, 19239-19245).

Acetonitriles↗

PubMed1994

Archaebacterial histone-like protein MC1 can exhibit a sequence-specific binding to DNA.

The binding of MC1 protein, the major chromosomal protein of the archaebacterium Methanosarcina sp. CHTI 55, to the region preceding the strongly expressed genes encoding methyl coenzyme reductase in a closely related micro-organism has been investigated. By gel retardation and DNAase I footprinting assays, we identified a preferential binding sequence in an open reading frame of unknown function. The large area of DNA protected against DNAase I is interrupted by a strong cleavage enhancement site on each strand. By circular permutation assays, we showed that the DNA bends upon MC1 binding. Furthermore we observed that the presence of a sequence outside the binding site can induce an unusual electrophoretic behaviour in some complexes.

Archaeal Proteins↗

PubMed1994

Fluorescence study on the non-specific binding of cyclic-AMP receptor protein to DNA: effect of pH.

The binding of the cyclic-AMP receptor protein (CRP) of Escherichia coli to a non-specific DNA fragment of 46 base pairs has been studied using fluorescence spectroscopy. The equilibrium binding constant was found to be several orders of magnitude lower than in the specific binding to a DNA fragment of the same size. The salt dependence of the equilibrium binding constant indicates that the CRP makes an identical number (8) of ion pairs to this non-specific DNA fragment in the presence and absence of cAMP. This number is larger than that previously found in the specific binding process. The effect of pH on the non-specific binding was investigated. The number of ion pairs does not vary between pH 6 and 8. From the variation of the binding constant with pH it was deduced that two histidines are involved in the binding in the absence of cAMP. These are most probably the histidines 199 of each subunit. In the presence of cAMP, only one histidine participates in the binding process, indicating an asymmetric interaction between the two subunits of the CRP and the DNA.

Cyclic AMP↗

PubMed1994

Comparison of solution structure of free and complexed lac operator by molecular modelling with NMR constraints.

The structure difference between the free operator of the lac system d(GCTCACAAT).d(ATTGTGAGC) and the same operator complexed to the headpiece of the lac repressor has been investigated by 2-D-1H NMR spectroscopy in conjunction with molecular modelling in internal coordinates (JUMNA). The free and complexed operator adopt both a right-handed B helical conformation, but a more detailed analysis of the conformational parameters using the Curves program shows striking differences in the groove geometries, the rises, the twists and the total bending.

Base Sequence↗

PubMed1994

Interaction of Hoechst 33258 with a DNA triple helix.

The interaction of Hoechst 33258 molecule, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix consisted of an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexa-ethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Circular dichroism spectroscopy demonstrates that Hoechst 33258 can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment slightly different in double-stranded and in triple-stranded structure. The affinity to the triple stranded structure is found smaller than to the double stranded one. Thermal denaturation experiments demonstrate that Hoechst 33258 destabilizes the triplex whereas it stabilizes the duplex.

Bisbenzimidazole↗

PubMed1994

Berenil complexation with a nucleic acid triple helix.

The interaction of berenil molecule, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12-X-(dT)12-X-(dT)12, where x is a hexaethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Circular dichroism spectroscopy demonstrates that berenil can bind to the triple helical structure. Spectral analysis shows that in the same ionic strength the drug bound to a double-stranded structure exhibits a conformation and an environment close to those observed in triple-stranded structure. The influence of the ionic strength on the interaction between the berenil molecule and the 36-mer is clearly demonstrated. We showed that when no NaCl salt is added in the buffer the triplex form of (dA)12-X-(dT)12-X-(dT)12 is stabilized by berenil whereas it is destabilized slightly by the dye when NaCl concentration is 1 M.

Base Sequence↗

PubMed1993

Stoichiometry of the binding of chromosomal protein MC1 from the archaebacterium, Methanosarcina spp. CHTI55, to DNA.

We have investigated the binding stoichiometry of the chromosomal MC1 protein on DNA using the gel retardation technique. Analysis of the distribution of the complex containing 0, 1, 2, 3 ... bound proteins shows that the protein MC1 interacts with the DNA as a monomer. Binding experiments with short DNA fragments of various lengths shows that the site size is 11 bp in length. These results are compared to those obtained with other chromosomal proteins including HU protein.

Archaeal Proteins↗

PubMed1992

Binding of netropsin to a DNA triple helix.

The interaction of netropsin, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide in a duplex form with a (dT)12 dangling arm. Circular dichroism spectroscopy demonstrates that netropsin can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment similar in double-stranded and in triple-stranded structure. However, the binding constant to the triple-stranded structure is found smaller than the binding constant to the double-stranded one. Thermal denaturation experiments demonstrate that netropsin destabilizes the triplex whereas it stabilizes the duplex.

Base Sequence↗

PubMed1992

Triple-helix formation by an oligonucleotide containing one (dA)12 and two (dT)12 sequences bridged by two hexaethylene glycol chains.

The triple-helix formation by the oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol group, was investigated by thermal denaturation analysis and circular dichroism spectroscopy. Thermal denaturation analysis showed that this single-stranded oligonucleotide is able to fold back on itself twice to give a triple helix at low temperature. Upon an increase in the temperature, two cooperative transitions were observed: formation of a double-stranded structure with a dangling x-(dT)12 extremity, then formation of a single-stranded coil structure. Due to the intramolecular character of the transition, the triplex is much more stable than that formed by the reference mixture (dA)12 + 2(dT)12. In 0.1 M NaCl, the triplex-to-coil transition occurred at about 30 degrees C whereas the duplex-to-coil was at about 60 degrees C. Upon an increase in the salt, the increase of temperature corresponding to the triplex-to-duplex transition was larger than that of the duplex-to-coil transition. MgCl2 showed higher efficiency than NaCl to promote triplex or duplex formation. The thermodynamic parameters delta H and delta S were determined at various ionic strengths for both transitions. Both the enthalpy change and entropy change associated with triplex-to-duplex transition (Hoogsteen base pairing) were smaller than those associated to the duplex-to-coil transition (Watson-Crick base pairing). When the ionic strength increased, the parameters -delta H and -delta S showed a very small decrease for the duplex-to-coil transition whereas a strong increase was observed with the triplex-to-duplex transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1992

Near ultraviolet circular dichroism study of the cyclic AMP receptor protein, its NH2-terminal domain and their interaction with cyclic AMP.

Circular dichroism in the near ultraviolet wavelength range was employed to examine conformational features of CRP (a dimer with a chain of 209 amino acids) and of its subtilisin core -alpha CRP- which retains the cAMP binding site (a dimer spanning the sequence 1-117). Binding of the ligand cAMP (allosteric activator), as well as cGMP was also investigated. The well resolved transitions could be assigned to the various classes of aromatic amino acid residues in the two proteins. In addition to signals which are attributable to the missing aromatic residues (Phe-136 and Tyr-206) the difference spectrum (CRP minus alpha CRP) shows a significant perturbation of a tryptophanyl contribution centred at 296 nm. From the available X-ray structure of the cAMP-CRP complex we are led to conclude that a conformational reorganisation takes place in the alpha CRP. A very large negative maximum is observed at 255 nm when cAMP binds to CRP and to alpha CRP. The maximum effect is observed in both cases at a ratio of one ligand bound per protomer. In the 280-300 nm wavelength range a smaller but significant perturbation affects specifically the spectra and reveals different cAMP-induced conformational changes in the two proteins. We propose that the major (255 nm) contribution to the perturbation spectrum of bound cAMP, and the qualitatively similar signal for cGMP, reflects an immobilisation of the sugar and adenine moieties of the bound ligand in an almost anti-conformation for both CRP and alpha CRP.

Bacterial Proteins↗

PubMed1992

Two-dimensional 1H, 15N NMR investigation of uniformly 15N-labeled lac repressor headpiece.

15N uniformly labeled lac repressor and lac repressor headpiece were prepared. 15N NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the 15N labeled N-terminal part of the protein is quite suitable for this type of study allowing future investigation of the specific interaction of the lac repressor headpiece with the lac operator. We report here the total assignment of proton 1H and nitrogen 15NH backbone resonances of this headpiece in the free state. Assignments of the 15N resonances of the protein were obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC nuclear Overhauser and relayed HMQC-HOHAHA spectroscopy. More than 80 per cent of residues were assigned by their 15NH(i)-N1H(i + 1) and 15NH(i)-N1H(i - 1) connectivities. Values of the 3JNH alpha splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed 15NH(i)-C beta H cross peaks and the 3JNH alpha coupling constants values are in agreement with the three alpha-helices previously described [Zuiderweg, E.R.P., Scheek, R.M., Boelens, R., van Gunsteren, W.F. and Kaptein, R., Biochimie 67, 707 (1985)]. The 3JNH alpha coupling constants can be now used for a more confident determination of the lac repressor headpiece. From these values it is shown that the geometry of the ends of the second and third alpha-helices exhibit deviation from the canonical alpha-helix structure. On the basis of NOEs and 3JNH alpha values, the geometry of the turn of the helix-turn-helix motif is discussed.

Amino Acid Sequence↗

PubMed1992

Specific binding of cyclic-AMP receptor protein to DNA. Effect of the sequence and of the introduction of a nick in the binding site.

The binding of Escherichia coli Cyclic AMP Receptor Protein (CRP) to several DNA fragments of about 45 base pairs, bearing the natural lactose or galactose sites, as well as several synthetic related sites, was investigated using fluorescence spectroscopy and gel retardation experiments. The salt dependence of the equilibrium binding constant indicates that CRP makes an identical number of ion pairs with the lac, lacL8 and gal sites although the binding constants are drastically different. However increasing the symmetry of the gal site leads to an increase of the number of ion pairs between the protein and the DNA. A single strand nick was introduced at the centre of a symmetrized gal site and this reduces the binding energy of CRP by about 0.6 Kcal. These results are discussed with respect to the bending constraints imposed on the DNA by the binding of CRP. The results are in agreement with the recently published crystal structure of the CRP complexed with DNA [Schutz, S.C., Shields, G.C. and Steitz, T.A., Science 253, 1001-1007 (1991)] showing that the 90 degrees bending of the DNA in the complex results from two kinks.

Base Composition↗

PubMed1991

The lac repressor and its N-terminal headpiece can bind a mini-operator containing a hairpin loop made of a hexaethylene glycol chain.

The binding of the lac repressor and the lac repressor N-terminal headpiece to a mini-operator with a hairpin loop made of a hexaethylene glycol chain was investigated using circular dichroism spectroscopy. The lac repressor's headpiece binds to the modified mini-operator with the same affinity as to a mini-operator of the same sequence without the hexaethylene glycol loop. The conformational effect due to the binding is not affected by the presence of the hexaethylene loop. It is also shown that the entire lac repressor binds to this modified mini-operator inducing a conformational change.

Base Sequence↗

PubMed1991

The chromosomal protein MC1 from the archaebacterium Methanosarcina sp. CHTI 55 induces DNA bending and supercoiling.

We have investigated the effect on the DNA structure of protein MC1, a basic and small polypeptide (Mr 10700) representing the major chromosomal protein in Methanosarcinaceae. The ability of protein MC1 to strongly favour cyclization upon polymerization of short DNA fragments by T4 DNA ligase indicates that protein MC1 mediates DNA bending. Several negatively supercoiled topoisomers of minicircles were obtained with DNA fragments of 203 and 146 bp, their distribution depends upon the amount of protein MC1 complexed with DNA. In addition, protein MC1 can induce a compaction of a nicked plasmid.

Archaeal Proteins↗