DNA interaction with human serum albumin studied by affinity capillary electrophoresis and FTIR spectroscopy

The question addressed in this study is how does the protein-DNA complexation affect the structure and dynamics of DNA and protein in aqueous solution. We examined the interaction of calf-thymus DNA with human serum albumin (HSA) in aqueous solution at physiological conditions, using constant DNA concentration of 12.5 mM (phosphate) and various HSA contents 0.25 to 2% or 0.04 to 0.3 mM. Affinity capillary electrophoresis and FTIR spectroscopic methods were used to determine the protein binding mode, the association constant, sequence preference, and the biopolymer secondary structural changes in the HSA-DNA complexes. Spectroscopic evidence showed two types of HSA-DNA complexes with strong binding of K(1) = 4.5 x 10(5) M(-1) and weak binding of K(2) = 6.10 x 10(4) M(-1). The two major binding sites were located on the G-C bases and the backbone PO(2) group. The protein-DNA interaction stabilizes the HSA secondary structure. A minor alteration of B-DNA structure was observed, while no major protein conformational changes occurred.     

Luminol–silver nitrate chemiluminescence enhancement induced by cobalt ferrite nanoparticles

CoFe₂O₄ nanoparticles (NPs) could stimulate the weak chemiluminescence (CL) system of luminol and AgNO₃, resulting in a strong CL emission. The UV–visible spectra, X‐ray photoelectron spectra and TEM images of the investigated system revealed that AgNO₃ was reduced by luminol to Ag in the presence of CoFe₂O₄ NPs and the formed Ag covered the surface of CoFe₂O₄ NPs, resulting in CoFe₂O₄–Ag core–shell nanoparticles. Investigation of the CL reaction kinetics demonstrated that the reaction among luminol, AgNO₃ and CoFe₂O₄ NPs was fast at the beginning and slowed down later. The CL spectra of the luminol ? AgNO₃ ? CoFe₂O₄ NPs system indicated that the luminophor was still an electronically excited 3‐aminophthalate anion. A CL mechanism has been postulated. When the CoFe₂O₄ NPs were injected into the mixture of luminol and AgNO₃, they catalyzed the reduction of AgNO₃ by luminol to produce luminol radicals and Ag, which immediately covered the CoFe₂O₄ NPs to form CoFe₂O₄–Ag core–shell nanoparticles, and the luminol radicals reacted with the dissolved oxygen, leading to a strong CL emission. With the continuous deposition of Ag on the surface of CoFe₂O₄ NPs, the catalytic activity of the core–shell nanoparticles was inhibited and a decrease in CL intensity was observed and also a slow growth of shell on the nanoparticles. Copyright © 2011 John Wiley & Sons, Ltd.     

Investigation of the interaction between melittin and dipalmitoylphosphatidylglycerol bilayers by vibrational spectroscopy.

Melittin is shown to affect the structure of the charged phospholipid dipalmitoylphosphatidylglycerol (DPPG). In the gel phase, the presence of melittin leads to (i) an increased lipid interchain vibrational coupling, (ii) a shift of the rectangular to hexagonal lipid packing transition toward low temperatures, (iii) a very small conformational disordering effect, (iv) a decrease of the polarity or hydrogen bonding capability of the lipid ester group surrounding, (v) an important decrease of the water content in the complexes where the remaining water has a more disordered structure than bulk water, and (vi) an interlamellar repeat distance of 79 A. All these observations are rationalized by the following model: adjacent bilayers of DPPG are bridged by tetramers of melittin through electrostatic interactions inducing surface charge neutralization and partial dehydration of the complexes. Melittin also affects the thermotropic behavior of DPPG. When a small amount of the toxin is present, its affinity for charged lipids is such that a phase separation occurs, the domains being stable enough to have their own gel to liquid-crystalline phase transition. In the fluid state, a deeper penetration into the lipid matrix is proposed based on the downshift of the phase transition and the low vibrational interchain coupling. This study brings out general features of cationic species/anionic lipid complexes. The charge neutralization leads to stronger interchain coupling, and electrostatic bridging of adjacent bilayers seems to be common. The hydrophobicity of the peptide is a key factor in the modulation of the gel to liquid-crystalline phase transition and in its insertion in the fluid lipid matrix.     

Study of interaction between Smad7 and DNA by single-molecule force spectroscopy

Smad7 is an antagonist of TGF-β signaling pathway and the mechanism of its inhibitory effect is of great interest. We recently found that Smad7 could function in the nucleus by binding to the DNA elements containing the minimal Smad binding element CAGA box. In this work, we further applied single-molecule force spectroscopy to study the DNA-binding property of Smad7. Smad7 showed similar binding strength to the oligonucleotides corresponding to the CAGA-containing activin responsive element (ARE) and the PAI-1 promoter, as that of Smad4. However, Smad7 also exhibited a binding activity to the mutant ARE with the CAGA sequence substituted, indicating its DNA-binding specificity is different from other Smads. Moreover, we demonstrated that the MH2 domain of Smad7 had a higher binding affinity to the DNA elements than the full-length Smad7, while the N-terminal domain exhibited an inhibitory effect.     

Investigation of the dark interaction between furocoumarins and DNA

The complexes between some furocoumarins and DNA have been studied using various physicochemical techniques. Flow-dichroism measurements data strongly support the intercalation of the planar furocoumarin molecules between two base pairs of duplex DNA. The equilibrium dialysis and spectrophotometric data show relatively low values of the association constants of the complexes and a small number of molecules able to intercalate in DNA, thus indicating that furocoumarins have a relatively low affinity for DNA in the complex formation. The biological and photobiological consequences connected with these results are discussed.The binding curves obtained using some polynucleotides and various DNA samples having different composition with regard to base pairs, have shown that the regions of the macromolecule having alternate sequences of purine and pyrimidine represent sites useful for intercalation. No preference has been observed for A-T or G-C.     

Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy.

Hsp70 chaperones assist protein folding through ATP-regulated transient association with substrates. Substrate binding by Hsp70 is controlled by DnaJ co-chaperones which stimulate Hsp70 to hydrolyze ATP and, consequently, to close its substrate binding cavity allowing trapping of substrates. We analyzed the interaction of the Escherichia coli Hsp70 homologue, DnaK, with DnaJ using surface plasmon resonance (SPR) spectroscopy. Resonance signals of complex kinetic characteristics were detected when DnaK was passed over a sensor chip with coupled DnaJ. This interaction was specific as it was not detected with a functionally defective DnaJ mutant protein, DnaJ259, that carries a mutation in the HPD signature motif of the conserved J-domain. Detectable DnaK-DnaJ interaction required ATP hydrolysis by DnaK and was competitively inhibited by chaperone substrates of DnaK. For DnaK mutant proteins with amino acid substitutions in the substrate binding cavity that affect substrate binding, the strength of detected interaction with DnaJ decreased proportionally with increased strength of the substrate binding defects. These findings indicate that the detected response signals resulted from DnaJ and ATP hydrolysis-dependent association of DnaJ as substrate for DnaK. Although not considered as physiologically relevant, this association allowed us to experimentally unravel the mechanism of DnaJ action. Accordingly, DnaJ stimulates ATP hydrolysis only after association of a substrate with the substrate binding cavity of DnaK. Further analysis revealed that this coupling mechanism required the J-domain of DnaJ and was also functional for natural DnaK substrates, and thus is central to the mechanism of action of the DnaK chaperone system.     

Comparative study of the interaction of fullerenol nanoparticles with eukaryotic and bacterial model membranes using solid-state NMR and FTIR spectroscopy

Native fullerene is notoriously insoluble in water and forms aggregates toxic to cell membranes, thus limiting its use in nanomedicine. In contrast, water-soluble fullerenol is compatible with biological systems and shows low in vivo toxicity on human cell lines. The interaction mechanism between these hydrophilic nanoparticles and biological membranes is however not well understood. Therefore, in this work, the effect of fullerenol on model eukaryotic and bacterial membranes was investigated using (31)P- and (2)H solid-state NMR as well as FTIR spectroscopy. DPPC/cholesterol and DPPC/DPPG bilayers were used to mimic eukaryotic and bacterial cell membranes, respectively. Our results show low affinity of fullerenol for DPPC/cholesterol bilayers but a clear interaction with model bacterial membranes. A preferential affinity of fullerenol for the anionic phospholipids DPPG in DPPC/DPPG membranes is also observed. Our data suggest that fullerenol remains at the water/bilayer interface of eukaryote-like membranes. They also indicate that the presence of a polar group such as DPPG's hydroxyl moiety at the bilayer surface plays a key role in the interaction of fullerenol with membranes. Hydrogen bonding of fullerenol nanoparticles with DPPGs' OH groups is most likely responsible for inducing lipid segregation in the lipid bilayer. Moreover, the location of the nanoparticles in the polar region of DPPG-rich regions appears to disturb the acyl chain packing and increase the membrane fluidity. The preferential interaction of fullerenol with lipids mostly found in bacterial membranes is of great interest for the design of new antibiotics.     

FTIR and UV spectroscopy studies of triplex formation between pyrimidine methoxyethylphosphoramidates alpha-oligodeoxynucleotides and ds DNA targets

The ability of non-ionic methoxyethylphosphoramidate (PNHME) alpha-oligodeoxynucleotides (ODNs), alpha dT(15) and alpha dCT dodecamer, to form triplexes with their double-stranded DNA targets was evaluated. Thermal stability of the formed complexes was studied by UV thermal denaturation and the data showed that these PNHME alpha-ODNs formed much more stable triplexes than phosphodiester (PO) beta-ODNs did (Delta Tm = + 20 degrees C for alpha dCT PNHME). In addition, FTIR spectroscopy was used to determine the base pairing and the strand orientations of the triplexes formed by alpha dT(15) PNHME compared to phosphodiester ODNs with beta or alpha anomeric configuration. While beta dT(15) PO failed to form a triplex with a long beta dA(n) x beta dT(n) duplex, the Tm of the Hoogsteen part of the triplex formed by alpha dT(15) PNHME reached 40 degrees C. Moreover alpha dT(15) PNHME displaced the beta dT(15) strand of a shorter beta dA(15) x beta dT(15) duplex. The alpha dCT PNHME and alpha dT(15) PNHME third strands were found antiparallel in contrast to alpha dT(15) PO which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides alpha dT and alpha dC combining both replacements might contribute to the improve stability of the triplexes.     

Investigation of DNA structural changes by infrared spectroscopy

DNA-oriented samples of various origins were studied under different conditions of humidiity and sodium chloride content by means of infrared spectroscopy. (1) Oriented DNA (M. Lysodeikticus, E. coli, calf thymus and salmon sperm) films at 3–4% sodium chloride yield polarized spectra which show drastic changes at relative humidities (r.h.) between 94% and 0% indicative of conformational changes: B form → a form → disordered form The measurements of the infrared dichroism at frequencies of about 1230 cm^?1 and at about 1090 cm^?1 allow one to determine the orientation of the phosphate group, whereas the measurements at 1710 cm^?1 characterize the base orientation. At humidities higher than 90% r.h. (B form) the bisector of OPO forms an angle of 70° relative to the helix axis, whereas at lower humidities, between 75% and 50% r.h. (A form) a rotation to about 45° is observed. Simultaneously, the 0—0 line of phosphate group changes its orientation from 55° to 65° to the helix when B → A transition takes place. The results are in general agreement with that of X-ray diffraction and allow one to determine the orientation of the phosphate group with greater precision. (2) The B–A conformational change is not observed for satellite DNA, isolated from Cancer pagurus, of which the guanine + cytosine content is below 5%. As a function of decreasing humidities, one observes the transition: B form → disordered form A diagram of conformational changes of DNA's as a function of base composition and of r.h., suggests that B–A transition will occur for DNA of relatively higher G + C content, whereas for high (A + T) content, base sequence may be of importance. The B–A transition is prevented in DNA at a relatively high or very low sodium chloride content.     

DNA protection by aminothiols: study of the cysteamine - DNA interaction by vibrational spectroscopy

Infrared linear dichroism measurements and Roman scattering spectra show that the cysteamine molecule binds strongly to the DNA stabilizing the double helix in a B geometry conformation. The B→A conformational transition is not observed for a cysteamine/DNA ratio of one cysteamine molecule per two phosphate sites. No evidence of interaction has been found between the radioprotector and the DNA bases. A model is proposed in which the cysteamine molecule is bound by its two ends through electrostatic interaction to two consecutive phosphate groups along the same DNA strand.     

Characterization of interaction between DNA and 4',6-diamidino-2-phenylindole by optical spectroscopy

We have examined the interaction between 4',6-diamidino-2-phenylindole (DAPI) and DNA using flow linear dichroism (LD), circular dichroism (CD), and fluorescence techniques. We show the presence of two spectroscopically distinct binding sites at low binding ratios with saturation values of 0.025 and 0.17, respectively. In both sites DAPI is bound with its long axis approximately parallel to the grooves of the DNA helix. Resolution of CD spectra shows that an exciton component is present at higher binding ratios, which we attribute to the interaction of two accidentally close-lying DAPI molecules. We also find evidence that DAPI, at least in the high-affinity site, binds preferentially to AT-rich regions. From the spectroscopic results, supported by structural considerations, we can completely exclude that DAPI is bound to DNA by intercalation. Binding geometries and site densities are consistent with a location of DAPI in the grooves of DNA, with the high-affinity site most probably in the minor groove.     

FTIR and UV spectroscopy studies of triplex formation between alpha-oligonucleotides with non-ionic phoshoramidate linkages and DNA targets

The triplexes formed by pyrimidine alpha-oligodeoxynucleotides, 15mers alpha dT(15) or 12mers alpha dCT having dimethoxyethyl (PNHdiME), morpholino (PMOR) or propyl (PNHPr) non-ionic phosphoramidate linkages with DNA duplex targets have been investigated by UV and FTIR spectroscopy. Due to the decrease in the electrostatic repulsion between partner strands of identical lengths all modifications result in triplexes more stable than those formed with unmodified phosphodiester beta-oligodeoxynucleotides (beta-ODNs). Among the alpha-ODN third strands having C and T bases and non-ionic phosphoramidate linkages (alpha dCTPN) the most efficient modification is (PNHdiME). The enhanced third strand stability of the alpha dCTPN obtained as diastereoisomeric mixtures is attenuated by the steric hindrance of the PMOR linkages or by the hydrophobicity of the PNHPr linkages. All alpha dCTPN strands form triplexes even at neutral pH. In the most favorable case (PNHdiME), we show by FTIR spectroscopy that the triplex formed at pH 7 is held by Hoogsteen T*A.T triplets and in addition by an hydrogen bond between O6 of G and C of the third strand (Tm = 30 degrees C). The detection of protonated cytosines is correlated at pH 6 with a high stabilization of the triplex (Tm = 65 degrees C). While unfavorable steric effects are overcome with alpha anomers, the limitation of the pH dependence is not completely suppressed. Different triplexes are evidenced for non pH dependent phosphoramidate alpha-thymidilate strands (alpha dT(15)PN) interacting with a target duplex of identical length. At low ionic strength and DNA concentration we observe the binding to beta dA(15) either of alpha dT(15)PN as duplex strand and beta dT(15) as third strand, or of two hydrophobic alpha dT(15)PNHPr strands. An increase in the DNA and counterion concentration stabilizes the anionic target duplex and then the alpha dT(15)PN binds as Hoogsteen third strand.     

FTIR spectroscopy of the complex between pharaonis phoborhodopsin and its transducer protein

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psRII) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR activates the cognate transducer protein, pHtrII, upon absorption of light. ppR and pHtrII form a tight 2:2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. In this paper, we studied the influence of pHtrII on the structural changes occurring upon retinal photoisomerization in ppR by means of low-temperature FTIR spectroscopy. We trapped the K intermediate at 77 K and compared the ppR(K) minus ppR spectra in the absence and presence of pHtrII. There are no differences in the X-D stretching vibrations (2700-1900 cm(-1)) caused by presence of pHtrII. This result indicates that the hydrogen-bonding network in the Schiff base region is not altered by interaction with pHtrII, which is consistent with the same absorption spectrum of ppR with or without pHtrII. In contrast, the ppR(K) minus ppR infrared difference spectra are clearly influenced by the presence of pHtrII in amide-I (1680-1640 cm(-1)) and amide-A (3350-3250 cm(-1)) vibrations. The identical spectra for the complex of the unlabeled ppR and (13)C- or (15)N-labeled pHtrII indicate that the observed structural changes for the peptide backbone originate from ppR only and are altered by retinal photoisomerization. The changes do not come from pHtrII, implying that the light signal is not transmitted to pHtrII in ppR(K). In addition, we observed D(2)O-insensitive bands at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII, which presumably originate from an X-H stretch of an amino acid side chain inside the protein.     

Insights into the binding interaction between copper ferrite nanoparticles and bovine serum albumin: An effect on protein conformation and activity

The binding affinity between bovine serum albumin (BSA) and copper ferrite (CuFe₂O₄) nanoparticles in terms of conformation, stability and activity of protein was studied using various spectroscopic methods. The quenching involved in BSA–CuFe₂O₄ NP interaction was static quenching as analysed by different techniques (steady‐state and time‐resolved fluorescence along with temperature‐dependent fluorescence measurements). Among all types of possible interactions, it was revealed that the major binding forces were van der Waals interaction and hydrogen bonding, which were explored from negative values of enthalpy change (?H = ?193.85 kJ mol^?1) and entropy change (?S = ?588.88 J mol^?1 K^?1). Additionally, synchronous, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy measurements confirmed the conformational changes in BSA upon the addition of CuFe₂O₄ NP. Furthermore, thermal denaturation observations were consistent with the circular dichroism results. The interaction of CuFe₂O₄ NP with BSA decreased the esterase activity in the BSA assay, revealing the affinity of copper ferrite towards the active site of BSA.     

Investigation into the acyl chain packing of endotoxins and phospholipids under near physiological conditions by WAXS and FTIR spectroscopy

The acyl chain packing of various endotoxins and phospholipids was monitored via the main wide-angle reflection between 0.410 and 0.460 nm by wide-angle X-ray scattering (WAXS) and via the absorption band of the symmetric stretching vibration of the methylene groups v(s)(CH(2)) around 2849 to 2853 cm(-1) by Fourier-transform infrared spectroscopy. The lipids investigated included various rough mutant (R) and smooth form (S) lipopolysaccharides (LPS) differing in the length of the sugar portion, lipid A, the "endotoxic principle" of LPS, and various saturated and unsaturated phospholipids with different head groups under a near physiological (>/=85%) water content. The packing density of the saturated endotoxin acyl chains is lower than those of saturated phospholipids but similar to those of monounsaturated phospholipids, each in the gel phase. The hydrophobic moiety of endotoxins thus exhibits significant conformational disorder already in the gel phase. The acyl chain packing of the endotoxins decreases with increasing length of the sugar chain lengths, which seems to be relevant to the observed differences in biological activity. For Re-LPS with different counterions (salt forms), in the presence of externally added cations or at reduced water content (50%), no change of the acyl chain packing density is deduced in the X-ray data, although the FT-IR data indicate its increase. The position of the v(s)(CH(2)) vibration is, thus, only a relative measure of lipid order, in particular when lipids with different head groups and in the presence of external agents are compared.     

Investigation on the interaction of cefpirome sulfate with lysozyme by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy

The reaction mechanism of cefpirome sulfate with lysozyme at different temperatures (298, 310 and 318 K) was investigated using fluorescence quenching and synchronous fluorescence spectroscopy under simulated physiological conditions. The results clearly demonstrated that cefpirome sulfate caused strong quenching of the fluorescence of lysozyme by a static quenching mechanism. The binding constants obtained using the above methods were of the same order of magnitude and very similar. Static electric forces played a key role in the interaction between cefpirome sulfate and lysozyme, and the number of binding sites in the interaction was close to 1. The values of Hill's coefficients were > 1, indicating that drugs or proteins showed a very weakly positive cooperativity in the system. In addition, the conclusions obtained from the two methods using the same equation were consistent. The results indicated that synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the fluorescence quenching method. In addition, the effect of cefpirome sulfate on the secondary structure of lysozyme was analyzed using circular dichroism spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Study by optical spectroscopy and molecular dynamics of the interaction of acridine-spermine conjugate with DNA

We report a spectroscopic and theoretical study of the interaction between double-stranded oligonucleotides containing either adenine-thymine or guanine-cytosine alternating sequences and N1-(Acridin-9-ylcarbonyl)-1,5,9,14,18-pentazaoctadecane, or ASC, which is formed by the covalent bonding of spermine and 9-amidoacridine moieties via a trimethylene chain. Solutions containing the oligonucleotides and the conjugate at different molar ratios were studied using complementary spectroscopic techniques, including electronic absorption, fluorescence emission, circular dichroism, and Raman spectroscopy. The spectroscopical properties of ASC at both the vibrational and the electronic levels were described by means of ab initio quantum-chemical calculations on 9-amidoacridine, used as a model compound. Molecular dynamics calculations, based on the QM/MM methodology, were also performed using previously docked structures of two oligonucleotide-ASC complexes containing the A-T and the G-C sequence. Our data, taken all together, allowed us to demonstrate that conjugation of spermine to acridine modulates and gives additional properties to the interaction of the latter with DNA. As the ASC molecule has a high affinity by the polyamine transport system, these results are promising for their application in the development of new anti-tumour drugs.     

Protein and DNA residue orientations in the filamentous virus Pf1 determined by polarized Raman and polarized FTIR spectroscopy

The Pseudomonas bacteriophage Pf1 is a long ( approximately 2000 nm) and thin ( approximately 6.5 nm) filament consisting of a covalently closed, single-stranded DNA genome of 7349 nucleotides coated by 7350 copies of a 46-residue alpha-helical subunit. The coat subunits are arranged as a superhelix of C(1)()S(5.4)() symmetry (class II). Polarized Raman and polarized FTIR spectroscopy of oriented Pf1 fibers show that the packaged single-stranded DNA genome is ordered specifically with respect to the capsid superhelix. Bases are nonrandomly arranged along the capsid interior, deoxynucleosides are uniformly in the C2'-endo/anti conformation, and the average DNA phosphodioxy group (PO(2)(-)) is oriented so that the line connecting the oxygen atoms (O.O) forms an angle of 71 degrees +/- 5 degrees with the virion axis. Raman and infrared amide band polarizations show that the subunit alpha-helix axis is inclined at an average angle of 16 degrees +/- 4 degrees with respect to the virion axis. The alpha-helical symmetry of the capsid subunit is remarkably rigorous, resulting in splitting of Raman-active helix vibrational modes at 351, 445 and 1026 cm(-)(1) into apparent A-type and E(2)()-type symmetry pairs. The subunit tyrosines (Tyr 25 and Tyr 40) are oriented with phenoxyl rings packed relatively close to parallel to the virion axis. The Tyr 25 and Tyr 40 orientations of Pf1 are surprisingly close to those observed for Tyr 21 and Tyr 24 of the Ff virion (C(5)()S(2)() symmetry, class I), suggesting a preferred tyrosyl side chain conformation in packed alpha-helical subunits, irrespective of capsid symmetry. The polarized Raman spectra also provide information on the orientations of subunit alanine, valine, leucine and isoleucine side chains of the Pf1 virion.