Thermodynamic basis of the α-helix and DNA duplex.

Analysis of calorimetric and crystallographic information shows that the α-helix is maintained not only by the hydrogen bonds between its polar peptide groups, as originally supposed, but also by van der Waals interactions between tightly packed apolar groups in the interior of the helix. These apolar contacts are responsible for about 60% of the forces stabilizing the folded conformation of the α-helix and their exposure to water on unfolding results in the observed heat capacity increment, i.e. the temperature dependence of the melting enthalpy. The folding process is also favoured by an entropy increase resulting from the release of water from the peptide groups. A similar situation holds for the DNA double helix: calorimetry shows that the hydrogen bonding between conjugate base pairs provides a purely entropic contribution of about 40% to the Gibbs energy while the enthalpic van der Waals interactions between the tightly packed apolar parts of the base pairs provide the remaining 60%. Despite very different structures, the thermodynamic basis of α-helix and B-form duplex stability are strikingly similar. The general conclusion follows that the stability of protein folds is primarily dependent on internal atomic close contacts rather than the hydrogen bonds they contain.

SYBR Green I: fluorescence properties and interaction with DNA.

In this study, we have investigated the fluorescence properties of SYBR Green I (SG) dye and its interaction with double-stranded DNA (dsDNA). SG/dsDNA complexes were studied using various spectroscopic techniques, including fluorescence resonance energy transfer and time-resolved fluorescence techniques. It is shown that SG quenching in the free state has an intrinsic intramolecular origin; thus, the observed >1,000-fold SG fluorescence enhancement in complex with DNA can be explained by a dampening of its intra-molecular motions. Analysis of the obtained SG/DNA binding isotherms in solutions of different ionic strength and of SG/DNA association in the presence of a DNA minor groove binder, Hoechst 33258, revealed multiple modes of interaction of SG inner groups with DNA. In addition to interaction within the DNA minor groove, both intercalation between base pairs and stabilization of the electrostatic SG/DNA complex contributed to increased SG affinity to double-stranded DNA. We show that both fluorescence and the excited state lifetime of SG dramatically increase in viscous solvents, demonstrating an approximate 200-fold enhancement in 100 % glycerol, compared to water, which also makes SG a prospective fluorescent viscosity probe. A proposed structural model of the SG/DNA complex is compared and discussed with results recently reported for the closely related PicoGreen chromophore.

Excitation volumetric effects (EVE) in metal-enhanced fluorescence.

Metal-Enhanced Fluorescence (MEF) effects from different density silver island films (SiFs) and the effects of far-field excitation irradiance on the observed enhancement of fluorescence were studied. It is shown that MEF non-linearly depends on silver nanoparticle (NP) size/density, reaching a maximum value for SiFs made at a deposition time (DT) of ∼5 minutes, i.e. just before SiFs become continuous. Numerical simulations of the silver-islands growing on glass revealed that the near-field magnitude depends non-linearly on size and interparticle distance exhibiting dramatic enhancement at ∼10 nm distance between the NPs. In addition, a remarkable effect of modulation in MEF efficiency by far-field excitation irradiance has been observed, which can be correlated well with numerical simulations that show an excitation power volume dependence. The near-field volume changes non-linearly with far-field power. This unique observation has profound implications in MEF, which has rapidly emerged as a powerful tool in the biosciences and ultimately allows for tunable fluorescence enhancement factors.

Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding.

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.

Metal-enhanced PicoGreen fluorescence: application to fast and ultra-sensitive pg/ml DNA quantitation.

In this paper we provide both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which non-covalently binds dsDNA. It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. We present experimental data obtained for a PicoGreen (PG)/DNA quantitation assay, which is in complete agreement with the results of our theoretical analysis. Experimentally measured PG-fluorescence intensity vs DNA concentration functions were fitted by a derived analytical expression, in which parameters of PG binding to DNA and chromophore fluorescence properties were included. We show that silver nanoparticles significantly increase the ratio between the fluorescence of PG bound to DNA and free PG, due to the metal-enhanced fluorescence effect (MEF), which enhances the lower limit of detectability of DNA concentrations by several orders of magnitude. An additional order of magnitude increase of PG/DNA assay sensitivity (~1 pg/ml) can be achieved by decreasing the PG concentration. We show herein that the use of MEF substrates in surface assays has a profound effect on assay sensitivity.

Metal-enhanced PicoGreen fluorescence: application for double-stranded DNA quantification.

PicoGreen (PG) is a fluorescent probe for both double-stranded DNA (dsDNA) detection and quantification based on its ability to form a luminescent complex with dsDNA as compared with the free dye in solution. To expand the sensitivity of PG detection, we have studied the spectral properties of PG, both free and in complex with DNA in solution, when the fluorophore is in proximity to silver nanoparticles. We show that for a broad range of PG concentrations (20 pM-3.5 microM), it does not form dimers/oligomers and it exists in a monomeric state. On binding to DNA in the absence of silver, PG fluorescence increases approximately 1100-fold. Deposition of PG/DNA complex onto silver island films (SiFs) increases fluorescence approximately 7-fold due to the metal-enhanced fluorescence (MEF) effect, yielding fluorescence enhancement of 7700-fold as compared with the free dye on glass. In contrast to PG in complex with DNA, the free dye on SiFs demonstrates a decrease in brightness approximately 5-fold. Therefore, the total enhancement of PG on binding to DNA on silver reaches a value of approximately 38,000 as compared with free PG on SiFs. Consequently, the metal-enhanced detection of PG fluorescence is likely to find important utility for amplified dsDNA quantification.

Stability and DNA-binding ability of the bZIP dimers formed by the ATF-2 and c-Jun transcription factors.

The dimer formed by the ATF-2 and c-Jun transcription factors is one of the main components of the human interferon-beta enhanceosome. Although these two transcription factors are able to form two homodimers and one heterodimer, it is mainly the heterodimer that participates in the formation of this enhanceosome, binding specifically to the positive regulatory domain IV (PRDIV) site of the enhancer DNA. To understand this surprising advantage of the heterodimer, we investigated the association of these transcription factors using fragments containing the basic DNA-recognition segment and the basic leucine zipper domain (bZIP). It was found that the probability of forming the hetero-bZIP significantly exceeds the probability of forming homo-bZIPs, and that the hetero-bZIP interacts more strongly with the PRDIV site of the interferon-beta enhancer, especially in the orientation that places the folded ATF-2 basic segment in the upstream half of this asymmetric site. The effect of salt on the formation of the ATF-2/c-Jun dimer and on its ability to bind the target PRDIV site showed that electrostatic interactions between the charged groups of these proteins and with DNA play an essential role in the formation of the asymmetric ATF-2/c-Jun/PRDIV complex.

DNA hydration studied by pressure perturbation scanning microcalorimetry.

Pressure perturbation differential scanning calorimetry was used to determine thermal expansion coefficients and thus temperature-induced volume changes of DNA duplexes differing in their GC/AT content. It was shown that the temperature-induced unfolding of the DNA duplexes proceeds with a significant increase of the thermal expansion coefficient and the partial volume of the DNA. Unusually, large temperature-induced changes in the partial volume were observed for an AT-rich dodecamer, a finding consistent with previous crystallographic studies showing the presence of highly ordered water molecules hydrating the minor groove of such duplexes. The data show that the density of this ordered water is substantially higher than that of the bulk water. This ordered water cannot, therefore, be equated to ice at normal pressures but it thermodynamically resembles ice formed at high pressures.

Metal-Enhanced Fluorescence (MEF): Physical Characterization of Siver-Island Films and Exploring Sample Geometries.

In this study we have analyzed metal-enhanced fluorescence (MEF) effects from different density silver island films (SiFs) and the effects of sample geometry on the observed enhancement of fluorescence (EF). It is shown that silver islands grow exponentially with SiF deposition time (DT<7min), optical density of SiFs almost linearly depends on DT; electrical conductivity is zero. At DT>7 min, silver islands merge, exhibiting a sharp increase in electrical conductivity. It has been shown that the newly proposed SiF-Glass sample geometry exhibits higher EF values than the commonly used in MEF studies SiF-SiF sample geometry. The SiF-Glass geometry demonstrates high sensitivity for surface immunoassays, a growing application of metal-enhanced fluorescence.

Assembling the human IFN-beta enhanceosome in solution.

Assembly of interferon-beta enhanceosome from its individual protein components and of enhancer DNA has been studied in solution using a combination of fluorescence anisotropy, microcalorimetry, and CD titration. It was shown that the enhancer binds only one full-length phosphomimetic IRF-3 dimer at the PRDIII-PRDI sites, and this binding does not exhibit cooperativity with binding of the ATF-2/c-Jun bZIP (leucine zipper dimer with basic DNA recognition segments) heterodimer at the PRDIV site. The orientation of the bZIP pair is, therefore, not determined by the presence of the IRF-3 dimer, but is predetermined by the asymmetry of the PRDIV site. In contrast, bound IRF-3 dimer interacts strongly with the NF-kappaB (p50/p65) heterodimer bound at the neighboring PRDII site. The orientation of bound NF-kappaB is also predetermined by the asymmetry of the PRDII site and is the opposite of that found in the crystal structure. The HMG-I/Y protein, proposed as orchestrating enhanceosome assembly, interacts specifically with the PRDII site of the interferon-beta enhancer by inserting its DNA-binding segments (AT hooks) into the minor groove, resulting in a significant increase in NF-kappaB binding affinity for the major groove of this site.

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5.

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.

The energetics of HMG box interactions with DNA. Thermodynamic description of the box from mouse Sox-5.

The structural energetics of the HMG box from the DNA-binding protein mouse Sox-5 were examined calorimetrically. It was found that this box, notwithstanding its small size (molecular mass about 10 kDa), does not behave as a single cooperative unit and, on heating, the box reversibly unfolds in two separate stages. The first transition (tt approximately 34 degrees C) involves about 40% of the total enthalpy and the second (tt approximately 46 degrees C) the remainder. Both transitions proceed with significant heat capacity increment, showing that they are associated with the unfolding of two sub-domains having non-polar cores. According to heat capacity, ellipticity, fluorescence and NMR criteria, this HMG box is in a fully compact native state only below 5 degrees C. HMG boxes consist of two approximately orthogonal wings: the minor wing comprises helix 3 and its associated antiparallel N-terminal strand, whilst the major wing is composed of helices I and II. Analysis of the fluorescence and NMR spectra for this box obtained at different temperatures shows that the lower melting transition can be assigned to the minor wing and the upper transition to the major wing. Under physiological conditions (37 degrees C), the minor wing is considerably unfolded, whilst the major wing is essentially fully folded. DNA binding in vivo therefore involves refolding of the minor wing.

Mechanisms of stabilizing nucleosome structure. Study of dissociation of histone octamer from DNA.

The influence of ionic strength on DNA-histone and histone-histone interactions in reconstituted nucleosomes was studied by measuring the parameters of histone tyrosine fluorescence: fluorescence intensity and lambda(max) position. The first parameter is sensitive to histone-DNA interactions. The changes of the second one accrue due to hydrogen bond formation/disruption between tyrosines in the histone H2A-H2B dimer and the (H3-H4)2 tetramer. The simultaneous measurement of these parameters permits the recording of both the dissociation of histone complexes from DNA, as well as changes in histone-histone interactions. As ionic strength is increased, the H2A-H2B histone dimer dissociated first, followed by dissociation of the (H3-H4)2 tetramer [Yager, T.G., McMurray, C.T. and Van Holde, K.E. (1989) Biochemistry 28, 2271-2276]. The H2A-H2B dimer is dissociated in two stages: first, the ionic bonds with DNA were disrupted, followed by the dissociation of the histone dimer from the tetramer. And secondly, the disruption of dimer-tetramer specific H-bonds. It was established that the energy of electrostatic interactions of the histone dimer with DNA within the nucleosome is much less than the energy of interaction of the histone dimer with the tetramer.

[Molecular mechanisms of the damaging effect of laser radiation on DNA]. / Molekuliarnye mekhanizmy povrezhdaiushchego deistviia lazernogo izlucheniia na DNK.

The effects of DNA photoscission were studied in the presence of ethidium bromide (EB) and riboflavin (RF) (vitamin B2). Laser irradiation (337 nm) of DNA in the presence of EB and RF induces single- and double-strand scissions in the superhelical form of DNA pBR322 (registered as increasing content of its nicked and linear forms). Possibility of DNA scission by energy absorbed by a nonintercalating chromophore (riboflavin) was demonstrated for the first time. The dependence of DNA photoscission in complex with riboflavin on the energy density in the pulse suggests a nonlinear (two-quantum) character of the photoscission process. It was shown that for riboflavin the quantum yield of DNA photoscission exceeds several times the one for ethidium bromide.

[Spectroscopy of intermolecular interactions of a tyrosine chromophore. III. Classification of the state of tyrosine residues in proteins based on their electron spectra]. / Spektroskopiia mezhmolekuliarnykh vzaimodeistvii tirozinovogo khromofora. III. Klassifikatsiia sostoianiia ostatkov tirozina v belkakh po ikh élektronnym spektram.

Absorption and fluorescence spectra of some tyrosine-containing proteins were analysed. Comparison of the peculiarities of fluorescence and absorption of the tyrosine chromophore in the model compounds and proteins suggested a new classification of the states of tyrosine residues in proteins: I -- tyrosyls with hydrated OH-group (lambda mf approximately equal to 304 nm); II -- tyrosyls, whose hydroxyl group forms the hydrogen bond inside the protein in a hydrophobic surrounding or in the globular fold in structured water layer (lambda mf = 306-307 nm); III -- tyrosyls whose OH-group is deprotonated in the excited state (lambda mf approximately equal to 330-350 nm).

Rearrangements of chromatin structure during spermatogenesis of squid.

A stepwise replacement of somatic histones on sperm-specific proteins (we have termed them illexines I1 and I2) is found to occur during spermatogenesis of squid Illex argentinus [Kadura, S.N. and Khrapunov, S.N. (1988) Eur. J. Biochem. 175, 603-607]. The chromatin from nuclei of squid immature testes has a nucleosomal DNA repeat which corresponds to the nucleosomal repeat of calf thymus chromatin (195 +/- 5 bp). As spermatozoa become mature and illexine I2 accumulates in the chromatin, the nucleosomal structure of the latter disappears and chromatin compacting takes place. The chromatin DNA from squid spermatozoa is highly resistant to micrococcal nuclease action. Spectrophotometry and spectrofluorimetry were to establish that neither illexine I1 nor illexine I2 forms a globular structure in solution under any conditions studied. Illexine I2 (approx. 7 kDa) shows a high affinity to DNA and remains bound to it under conditions when complexes of illexine I1 (approx. 9 kDa) and salmine (approx. 4.5 kDa) with DNA completely dissociate. This fact, allowing for a similar content (about 75%) of arginine in illexine I2 and salmine, suggests high clustering of arginine residues in the composition of illexine I2. It is suggested that the initial stage of histone substitution with illexine I1, which has a more moderate affinity to DNA than illexine I2, prepares chromatin for the formation of a highly packed structure by illexine I2 during squid spermatogenesis.

[Theoretical study of structural transition in a nucleosome at low ionic strength]. / Teoreticheskoe issledovanie strukturnogo perekhoda v nukleosome pri nizkoi ionnoi sile.

The theoretical analysis of nucleosome stability at low ionic strength has been performed on the basis of consideration of different contributions to the free energy of compact state of the nucleosome DNA terminal regions. The proposed model explains: the fact of low-salt structural change; the transition point (approximately 1.7 mM NaCl) and width (approximately 1 mM); the shift of the transition to the higher salt concentrations in the case of histones tails removal by trypsin. According to the model the increase of electrostatic repulsion between neighbouring turns of DNA superhelix is the main cause of the unwinding of nucleosomal DNA terminal regions in the course of low-salt structural change. The interactions between histone (H2A-H2B) dimer and (H3-H4)2 tetramer provide the compact state of the nucleosomal DNA terminal regions. The existence of electrostatic interactions of nucleosomal DNA terminal regions with tetramer was suggested. These interactions can provide the compact state of nucleosomal DNA at physiological ionic strength even in the absence of (H2A-H2B) dimer.

[The nature of forces stabilizing nucleosome structure. Dissociation of histone octamers from DNA]. / Priroda sil, stabiliziruiushchikh strukturu nukleosomy. Issledovanie protsessa dissotsiatsii oktamera gistonov ot DNK.

We have used the measurements of the histone fluorescence parameters to study the influence of the ionic strength on histone-DNA and histone-histone interactions in reconstructed nucleosomes. The ionic strength increase lead to the two-stage nucleosome dissociation. The dimer H2A-H2B dissociates at the first stage and the tetramer (H3-H4)2 at the second one. The dimer H2A-H2B dissociation from nucleosome is a two-stage process also. The ionic bonds between (H2A-H2B) histone dimer and DNA break at first and then the dissociation of dimer from histone tetramer (H3-H4)2 occurs. According to the proposed model the dissociation accompanying a nucleosome "swelling" and an increase of DNA curvature radius. It was shown that the energy of electrostatic interactions between histone dimer and DNA is sufficiently less than the energy of dimer-tetramer interaction. We propose that the nucleosome DNA ends interact with the dimer and tetramer simultaneously. The calculated number (approximately 30 divided by 40) of ionic bonds between DNA and histone octamer globular part practically coincides with the number of exposed cationic groups on the surface of octamer globular head. On this basis we have assumed that the spatial distribution of these groups is precisely determined, which explains the high evolutionary conservatism of the histone primary structure.

[Structure of histone octamers in reconstituted polynucleosomes]. / Struktura oktamera gistonov v sostave rekonstruirovannykh polinukleosom.

The salt-dependent structural changes of the histone octamer in complex with high-molecular-weight DNA have been studied by fluorescent spectroscopy. Changes in both the spectra maximum position and anisotropy of the histone tyrosine fluorescence reveal structural transitions in nucleosome within the ranges of 0.5-3 mM and 20-30 mM NaCl. Comparison of the octamer fluorescent parameters in complex with DNA as well as in a free state permits to interpret the revealed structural transitions as a change in degree of contacts stability between (H2A-H2B) dimer and (H3-H4)2 tetramer. More pronounced conformational changes in histone octamer are observed under the conditions of polynucleosome fibers interaction within the range of physiological ionic strength (100-600 mM NaCl). As far as fluorescent parameters are concerned, the aforementioned changes are connected with entire destruction of (H2A-H2B) dimer specific contacts with (H3-H4)2 tetramer. The obtained results suggest the possibility of existence of different structural states of histone octamer in the chromatin composition including those which are quite dissimilar from the octamer structure in the 2M NaCl solution.

[Two structural forms of histone octamer]. / Dve strukturnye formy oktamera gistonov.

It has been found that histone octamer of calf thymus (H2A--H2B--H3--H4)2 can exist in two structural states--"loose" (2M NaCl) and "compact" one (4M NaCl). The compact state of the octamer is characterized by screening of part of tyrosyls for quenching effect of ions I-, longer relaxation time of tyrosyls, greater stability of histone H3 towards trypsinolysis, complete absence of interactions between histone H3 SH-groups and parachlormercuribenzoate.