Radiation damage to a DNA-binding protein. Combined circular dichroism and molecular dynamics simulation analysis.

The E. coli lactose operon, the paradigm of gene expression regulation systems, is the best model for studying the effect of radiation on such systems. The operon function requires the binding of a protein, the repressor, to a specific DNA sequence, the operator. We have previously shown that upon irradiation the repressor loses its operator binding ability. The main radiation-induced lesions of the headpiece have been identified by mass spectrometry. All tyrosine residues are oxidized into 3,4-dihydroxyphenylalanine (DOPA). In the present study we report a detailed characterization of the headpiece radiation-induced modification. An original approach combining circular dichroism measurements and the analysis of molecular dynamics simulation of headpieces bearing DOPA-s instead of tyrosines has been applied. The CD measurements reveal an irreversible modification of the headpiece structure and stability. The molecular dynamics simulation shows a loss of stability shown by an increase in internal dynamics and allows the estimation of the modifications due to tyrosine oxidation for each structural element of the protein. The changes in headpiece structure and stability can explain at least in part the radiation-induced loss of binding ability of the repressor to the operator. This conclusion should hold for all proteins containing radiosensitive amino acids in their DNA-binding site.

Stability of the lac repressor headpiece against thermal denaturation and tryptic hydrolysis.

The stability of the conformation of the lac repressor headpiece against thermal denaturation and tryptic hydrolysis has been studied by circular dichroism measurements. In both cases the stability depends strongly on the concentration of NaCl. This effect is larger than generally observed for proteins. The midpoint of the thermal denaturation curve (Tm) is shifted from about 37 degrees C in the absence of NaCl to about 68 degrees C in 1 M NaCl. After a first non-linear increase of the Tm with the NaCl concentration (up to about 0.2 M NaCl) the Tm varies linearly with the salt concentration. Assuming a two-state mechanism for the thermal denaturation, enthalpies of 30-36 kcal/mol have been determined. The decrease of the circular dichroism signal due to the tryptic cleavage follows pseudo first-order kinetics for all salt concentrations studied. The half-life time of hydrolysis increased by about 40-times from 2 mM to the highest NaCl concentration we have used (655 mM). Assuming that only the unfolded state of the headpiece is a good substrate for trypsin, the observed stabilization against proteolytic degradation may be explained by a shift of the unfolding equilibrium of the headpiece due to the salt, and a subsequent decrease of the concentration of the unfolded state. The unusual stabilization of the headpiece is discussed with respect to its positive charge and to its function to bind to DNA.

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.

Interaction between the cyclic AMP receptor protein and DNA. Conformational studies.

The binding of the cyclic adenosine 3',5' monophosphate receptor protein (CRP or CAP) of Escherichia coli to non-specific DNA and to a specific lac recognition sequence has been investigated by circular dichroism (c.d.) spectroscopy. The effect of cAMP and cGMP on the co-operative non-specific binding was also studied. For the non-specific binding in the absence of cAMP a c.d. change (decrease of the intensity of the positive band with a shift of its maximum to longer wavelength) indicates that the DNA undergoes a conformational change upon CRP binding. This change might reflect the formation of the solenoidal coil previously observed by electron microscopy. The amplitude of the c.d. change increases linearly with the degree of saturation of the DNA and does not depend on the size of the clusters of CRP bound. From the variation of the c.d. effect as a function of the ionic strength, the product K omega (K, the intrinsic binding constant and omega, the co-operativity parameter) could be determined. The number of ion pairs involved in complex formation between CRP and DNA was found to be six to seven. Experiments performed with several DNAs, including the alternating polymers poly[d(A-T)] and poly[d(G-C)], demonstrated that the conformational change does not depend on the DNA sequence. However, in the presence of cAMP the c.d. spectrum of the DNA shows only a small variation upon binding CRP. In contrast, in the presence of cGMP the conformational change of the DNA is similar to that observed when non-liganded CRP binds. For the specific lac operon binding, the c.d. change is different from those observed for non-specific binding in the presence or absence of cAMP. These results emphasize the high variability of the DNA structure upon binding the same protein.

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.

[Effect of substituents on photochemical and biological properties of 13,15-N-cycloimide derivatives of chlorin p6].

The effect of electron-accepting substituents in position 3 of the chlorine p6 macrocycle in neutral and carboxyl-containing negatively charged cycloimide derivatives of chlorin p6 (CIC) on the photochemical and biological properties of these photosensitizers was studied. A relationship between the structure and properties of CICs was analyzed on the basis of information on their photoinduced cytotoxicity, efficiency of the generation of reactive oxygen species, photostability, intracellular localization, quantitative parameters of accumulation in cells, and cellular pharmacokinetics. It was shown that these compounds can be used for the development of photosensitizers with intense light absorption at 740 nm, controlled intracellular localization, and a high photodynamic activity toward tumor cells.

Thermodynamic parameters of the binding of the tight-binding I12X86 lac repressor to operator and non-operator DNA.

The thermodynamic parameters delta H and delta S corresponding to the binding of the tight-binding double mutant lac repressor I12X86 with operator and non-operator DNA fragments were determined using the nitrocellulose filter binding assay. In both cases the binding processes are entropically driven and accompanied by an unfavorable enthalpy variation. The differences between these parameters and those previously reported for the wild type lac repressor show that the strategy adopted by the mutant to interact with DNA is highly different from that of the wild type repressor and suggest more hydrophobic contacts between the mutant and DNA.

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.

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.

Interaction of lac repressor with alternating poly d (A-T) and poly d (G-C). Circular dichroism studies.

The binding of lac repressor to poly d(A-T) and d(G-C) has been studied using circular dichroism. The results indicate that the binding induces the same conformational change of both polynucleotides and perturbs the same number of nucleic acid bases (28 bases). It is shown that in 0.1 M phosphate buffer the CD measurement can be used to determine the binding constant of lac repressor to poly d(A-T). Competition experiments performed at various salt concentrations show that the stronger interaction of lac repressor for poly d(A-T) than for poly d(G-C) is based on difference in the dissociation rate of the complexes whereas the association rate for both polymers are similar.

The interaction between rat plasma alpha 1 inhibitor 3 and chymotrypsin. A protease-protease inhibitor system which gives partially active complexes.

Complexation of chymotrypsin with rat alpha 1 inhibitor 3 (alpha 1I3) leads to a significant modification of circular dichroism (CD) spectra in the near ultraviolet. These spectra variations are due to structural changes of the inhibitor resulting from partial proteolysis. Taking advantage of this change in CD spectral properties a 1:1 stoichiometric ratio was determined for the interaction. However, when an excess of inhibitor was present in the mixture, a slow change of the CD spectrum was observed. This was shown to result from a partial proteolysis of native inhibitor identical to that occurring during complex formation. This hydrolysis was due to the remaining activity of the bound enzyme since no significant release of free enzyme from the complex was demonstrated. Addition of human alpha 1 proteinase inhibitor (alpha 1 Pi) to dissociate the complex resulted in the formation of an enzymatically inactive ternary complex which gave evidence for the stability of the alpha 1I3 chymotrypsin binding.

A circular dichroism study of the Turnip yellow mosaic virus-RNA.

We examined the circular dichroism spectra of intact Turnip yellow mosaic virus, freezed/thawed virus, empty capsid particles, and phenol extracted RNA. The circular dichroism signal of the empty capsid was found to contribute for less than 1% to the circular dichroism of the virus. Differences in the circular dichroism spectra indicate that TYMV-RNA exhibits different conformations when it is in situ in the virus, when it has been ejected by freezing/thawing and when it has been phenol extracted. Increase of the ionic strength up to 0.1 M NaCl led to conformational change of the RNA either freezed/thawed ejected or phenol extracted but not in situ in the capsid. Addition of spermidine (3 mM) induced a conformational change only for the phenol extracted RNA. These results are discussed with respect to the origin of the various conformational states of viral RNA.

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.

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.

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.

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).

Nucleic acids-protein interactions. Conformational changes induced by the binding of aromatic amines to polyadenylic acid.

The binding of Tryptamine, Serotonine, Phenylethylamine and Histamine to poly(A) in its single stranded form at pH 7 leads to a decrease of its circular dichroism (C.D) amplitude without any appreciable alteration of the shape of the C.D. spectrum. The magnitude of the effect depends on the size of the aromatic ring and decreases in the order : tryptamine greater than tyramine greater than phenylethylamine greather than histamine. A method is described which allows the calculation of association constants from C.D. data. The C.D. amplitude decreases linearly with concentration of bound molecules. Binding of aromatic amines to poly(A) leads to a change in the proton chemical shifts of both the amine and the poly(A) protons. Quantitative analysis of P.M.R. data demonstrates that the shifts of poly(A) protons are linearly related to the concentration of bound molecules.

Oligodeoxynucleotides covalently linked to intercalating agents: a new class of gene regulatory substances.

Oligodeoxynucleotides have been covalently linked to a 9-aminoacridine derivative via their 3'-phosphate group. Specific complexes are formed with the complementary sequence of the oligonucleotide. The stability is strongly increased due to intercalation of the acridine derivative. Absorption, fluorescence, nuclear magnetic resonance and circular dichroism have been used to characterize complex formation. The stability of the complexes depends on the length of the linker between the acridine derivative and the 3'-phosphate group of the oligonucleotide. Oligonucleotides covalently linked to an intercalating agent can be used to selectively control gene expression. Transcription initiation can be blocked when such an oligonucleotide binds to the transcribed strand in the open complex formed by E. coli RNA polymerase with the bla promoter. With some oligonucleotides, non-specific effects on transcription can be detected, most probably due to binding of the modified oligonucleotide to RNA polymerase. Translation of the messenger RNA from gene 32 of phage T4 can be prevented by using an oligonucleotide complementary to the sequence upstream from the Shine-Dalgarno sequence. Inhibition of translation does not occur in the absence of the intercalating agent covalently linked to the oligonucleotide nor with oligonucleotides which do not have a target sequence on the mRNA.

'Non-specific' binding of lac repressor of E. coli to a carcinogen-modified DNA.

The 'non-specific' binding of lac repressor to a N-hydroxyaminofluorene (N-OH-AF) modified DNA was studied using circular dichroism. The binding process was not altered by the modification of the DNA. A small increase of the binding constant was observed. These results are discussed with respect to the role of carcinogen modification in the protein-nucleic acid recognition phenomena.

Nonspecific binding of lac repressor to DNA. I. An absorption and circular dichroism study.

Nonspecific binding of lac repressor on DNA has been studied by absorption and circular dichroism (CD) spectroscopies. In a first step, the complex formation is accompanied by an absorption difference spectrum and a change of the CD signal of the DNA. The absorption difference spectrum is mainly due to a spectral change of the DNA. The variation of the CD signal has been analyzed according to a model calculation, which takes into account the fact that the excluded site is shorter than the perturbed site. We found that in this first step one repressor can bind every 14 +/- 2 base-pairs, whereas one repressor perturbs 22 +/- 2 base-pairs. In a second step, more repressor can bind on DNA, but without further change in the absorption and CD spectrum, indicating that another binding process occurs. The model calculation developed here is general for all binding processes inducing a perturbation over a length of DNA longer than that of the excluded site.