Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides

Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. DAPI decay at pH 7 was decomposed using two exponential components of 2.8 and 0.2 ns of lifetime values, respectively. The double exponential character of the decay was maintained over a large pH range. Phase- and modulation-resolved spectra, collected between 420 and 550 nm, have indicated at least two spectral components associated with the two lifetime values. This, plus the observation of the dependence of the emission spectrum on the excitation wavelength, suggests a lifetime heterogeneity originating from ground-state molecular conformers, partially affected by pH changes. DAPI complexed with natural polydeoxynucleotides retained most of the features of DAPI decay in solution, except for the value of the long lifetime component that was longer (approximately 4 ns) and the relative fractional fluorescence intensities of the two components that were inverted. AT polymers/DAPI complexes show single exponential decay. Solvent shielding when DAPI is bound to DNA changes the indole ring solvation and stabilizes the longer lifetime decay component. For poly(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence the different types of binding and the reliability of the fluorescence method to solve them.     

Structural properties of DNO investigated with pyrene excimer formation.

Four diamino acid-Nalpha-substituted oligopeptide (DNO) oligomers substituted with pyrenyl as photophysical probes were synthesized. The excimer formation and ground-state association of the pyrenyl groups were investigated by means of absorption and steady-state fluorescence spectroscopy together with time-resolved fluorescence techniques. The photophysical parameters obtained from the different derivatives reflect the secondary structural properties of the DNO backbones.     

The pH-dependent conformational transition of beta-lactoglobulin modulates the binding of protoporphyrin IX

We have investigated the interaction between PPIX and beta-lactoglobulin (beta-lg) as a function of the pH of the solution. beta-lg is a small globular protein (MW approximately 18 kDa) with a very well characterized structure that reveals several possible binding sites for ligands. The interaction with beta-lg affects the photophysical properties of PPIX. The shift of PPIX emission maximum, excitation maximum and the increase of the fluorescence intensity is an indicator that binding between the porphyrin and beta-lg occurs. The binding constant appears to be modulated by the pH of the solution. Spectroscopic measurements do not reveal any significant energy transfer between the Trp residues of beta-lg and PPIX, however, fluorescence anisotropy decay measurements confirm the binding and the modulation introduced by the pH of the solution. Since beta-lg has been shown to be stable within the range of pH adopted in our experiments (5.0-9.0), the results suggest that PPIX binds a site affected by the pH of the solution. Because of the crystallographic evidence an obvious site is near the aperture of the interior beta-barrel however an alternative (or concurrent) binding site may still be present.     

Time-resolved fluorescence and 1H NMR studies of tyrosine and tyrosine analogues: correlation of NMR-determined rotamer populations and fluorescence kinetics

The time-resolved fluorescence properties of phenol and straight-chained phenol derivatives and tyrosine and simple tyrosine derivatives are reported for the pH range below neutrality. Phenol and straight-chained phenol derivatives exhibit single exponential fluorescence decay kinetics in this pH range unless they have a titratable carboxyl group. If a carboxyl group is present, the data follow a two-state, ground-state, Henderson-Hasselbalch relationship. Tyrosine and its derivatives with a free carboxyl group display complex fluorescence decay behavior as a function of pH. The complex kinetics cannot be fully explained by titration of a carboxyl group; other ground-state processes are evident, especially since tyrosine analogues with a blocked carboxyl group are also multiexponential. The fluorescence kinetics can be explained by a ground-state rotamer model. Comparison of the preexponential weighting factors (amplitudes) of the fluorescence decay constants with the 1H NMR determined phenol side-chain rotamer populations shows that tyrosine derivatives with a blocked or protonated carboxyl group have at least one rotamer exchanging more slowly than the radiative and nonradiative rates, and the fluorescence data are consistent with a slow-exchange model for all three rotamers, the shortest fluorescence decay constant is associated with a rotamer where the carbonyl group can contact the phenol ring, and in the tyrosine zwitterion, either rotamer interconversion is fast and an average lifetime is seen or rotamer interconversion is slow and the individual fluorescence decay constants are similar.     

The pH-dependent conformational transition of β-lactoglobulin modulates the binding of protoporphyrin IX

We have investigated the interaction between PPIX and β-lactoglobulin (β-lg) as a function of the pH of the solution. β-lg is a small globular protein (MW ≈18 kDa) with a very well characterized structure that reveals several possible binding sites for ligands. The interaction with β-lg affects the photophysical properties of PPIX. The shift of PPIX emission maximum, excitation maximum and the increase of the fluorescence intensity is an indicator that binding between the porphyrin and β-lg occurs. The binding constant appears to be modulated by the pH of the solution. Spectroscopic measurements do not reveal any significant energy transfer between the Trp residues of β-lg and PPIX, however, fluorescence anisotropy decay measurements confirm the binding and the modulation introduced by the pH of the solution. Since β-lg has been shown to be stable within the range of pH adopted in our experiments (5.0–9.0), the results suggest that PPIX binds a site affected by the pH of the solution. Because of the crystallographic evidence an obvious site is near the aperture of the interior β-barrel however an alternative (or concurrent) binding site may still be present.     

Fluorescent complexes of DNA with DAPI 4′,6-diamidine-2-phenyl indole.2HCl or DCI 4′,6-dicarboxyamide-2-phenyl indole

4',6-Dioarboxyamide-2-phenyl indole (DCI), a non-ionic structural analogue of 4',6-diamidine-2-phenyl indole.2HCl (DAPI), was synthesized in order to verify the hypothesis of intercalation of both dyes into the DNA double helix.The influence of pH, viscosity, and different concentrations of SDS (sodium dodecylsulphate) or NaCl on the optical and fluorescent properties and the changes in thermal transition of both dye complexes with DNA confirm the affinity of the dyes to the double helix as well as their stabilizing influence on the secondary DNA structure.The results of binding studies, carried out by fluorescent methods have shown that the dyes are strongly bound to DNA, though the number of binding sites is small. According to the experimental data, the fluorescent properties of DAPI and DCI complexes with DNA are connected with the intercalating binding mechanism of these dyes. On the other hand, the eventual ionic or hydrogen bonds of dyes outside the DNA helix do not change noticeably their fluorescent properties.     

去辅基天青蛋白两种天然构象共存的热力学证据 ———差热温度扫描和圆二色的研究

天然态蛋白质能否在溶液中存在多种构象是一个有争议的问题 . 在前报道中已经鉴定出绿脓杆菌去辅基天青蛋白突变体 M121L 可以多种构象共存 . 用差热扫描量热和圆二色性的方法研究了野生型去辅基天青蛋白的热变性 . 结果表明在 pH 从 4.0 到 9.0 的范围内存在着两个摩尔热容最大值 . 较低温度下的去折叠反应在所研究 pH 范围内均部分可逆,而较高温度下的去折叠反应均不可逆 . 蛋白质去折叠的热容变化双峰用可相互转化的两种构象共存模型进行拟合 . 较低温度下能够去折叠的构象在 pH 4.0 时占 64% ,在 pH 9.0 时占 55%. 监测热变性过程中圆二色谱在 219 nm 处的信号变化也可以观测到两个独立的去折叠变化 . 信号变化的比值与在相同条件下差热扫描法测得的两种构象摩尔比一致 . 上述结果进一步支持了前文提出的去辅基天青蛋白在溶液中至少存在着两种构象的设想 .     

Tryptophan Conformations Associated with Partial Unfolding in Ribonuclease T1

The origin of the biexponential fluorescence decay of Trp in ribonuclease T1 under mildly destabilizing conditions, such as increased pH or temperature, or the presence of detergent, is still not understood. We have performed two extended replica-exchange molecular dynamics simulations to obtain a detailed representation of the native state at two protonation states corresponding to a high and low pH. At high pH, the appearance of partially unfolded states is evident. We found that this pH-induced destabilization originates from increased global repulsion as well as reduced local favorable electrostatic interactions and reduced H-bonding strength of His²⁷, His⁴⁰, and His⁹². At high pH, alternative tryptophan rotamers appear and are linked to a distorted environment of the tryptophan, which also acts as a separate source of ground-state heterogeneity. The total population of these alternative conformations agrees well with the amplitude of the experimentally observed secondary fluorescence lifetime.     

Structure and interactions of aggrecans: statistical thermodynamic approach

Weak polyelectrolytes tethered to cylindrical surfaces are investigated using a molecular theory. These polymers form a model system to describe the properties of aggrecan molecules, which is one of the main components of cartilage. We have studied the structural and thermodynamical properties of two interacting aggrecans with a molecular density functional theory that incorporates the acid-base equilibrium as well as the molecular properties: including conformations, size, shape, and charge distribution of all molecular species. The effect of acidity and salt concentration on the behavior is explored in detail. The repulsive interactions between two cylindrical-shaped aggrecans are strongly influenced by both the salt concentration and the pH. With increasing acidity, the polyelectrolytes of the aggrecan acquire charge and with decreasing salt concentration those charges become less screened. Consequently the interactions increase in size and range with increasing acidity and decreasing salt concentration. The size and range of the forces offers a possible explanation to the aggregation behavior of aggrecans and for their ability to resist compressive forces in cartilage. Likewise, the interdigitation of two aggrecan molecules is strongly affected by the salt concentration as well as the pH. With increasing pH, the number of charges increases, causing the repulsions between the polymers to increase, leading to a lower interdigitation of the two cylindrical polymer layers of the aggrecan molecules. The low interdigitation in charged polyelectrolytes layers provides an explanation for the good lubrication properties of polyelectrolyte layers in general and cartilage in particular.     

Structural states and transitions of carp hemoglobin.

The wide ligand affinity range previously observed for carp hemoglobin is bounded at both extremes by regions of constant affinity. Within these regions, pH, organic phosphates, and the extent of ligand binding have no effect on the measured affinity and the cooperativity of ligand binding is greatly reduced or absent. The rates of CO recombination to fully and partially unliganded carp hemoglobin, under various organic phosphate and pH conditions, are shown to reflect this behavior. Constant kinetic rates are seen to directly correspond to the regions of constant affinity. Therefore, these are taken to be single protein conformations, one of high and one of low ligand affinity. In the simplest view, these conformations represent the R and T states of a two-state model, and most of the properties of carp hemoglobin are explained quite well within this framework. Increases in either hydrogen or phosphate ion concentrations favor the stabilization of the low affinity structure of even fully liganded carp hemoglobin. We have studied the structural transition from high to low affinity by monitoring the absorption spectra of carp hemoglobins at constant pH as a function of organic phosphate concentration. We find that different spectra are induced in both carp methemoglobin and cyanomethemoglobin by inositol hexaphosphate addition. Furthermore, the dependence of the magnitude of the spectral changes on pH and organic phosphate concentration is the close agreement with that predicted from studies of the ligand binding properties of the molecule.     

Peptide-sandwiched protoporphyrin compounds mimicking hemoprotein structures in solution

A series of covalently bound peptide-protoporphyrin-peptide compounds, also carrying naphthalene (N) to allow a photophysical investigation, were synthesized. Their general formula is P(nN)(2), where P refers to protoporphyrin IX, and n to the number of amino acids in the sequence Boc-Leu-Leu-Lys-(Ala)(x) -Leu-Leu-Lys-OtBu of each backbone chain (x = 0-3; n = x + 6). Their structural features in methanol solution were investigated by ir and CD spectra, and by steady-state and time resolved fluorescence experiments as well. The ir spectra indicate that intramolecularly H-bonded conformations form, and CD data in both methanol and water-methanol mixture suggest the presence of alpha-helix structure. Quenching of excited naphthalene takes place by electronic energy transfer from singlet N* to P ground state. Fluorescence decays coupled with molecular mechanics calculations indicate that two conformers for each dimeric peptide are the major contributors to the observed phenomena. These conformers are characterized by a globular, protein-like structure, where the protoporphyrin resides in a central pocket, while the two N groups are externally situated. Of the four N linkages in the two conformers, three of them attain a very similar steric arrangement around the central P molecule, in terms of both center-to-center distance and mutual orientation, while the fourth experiences a different steric disposition as compared to the others. Experimental photophysical parameters satisfactorily compare with those obtained by theoretical calculations, within the F?rster mechanism for long-range energy transfer, only when the mutual orientation of the chromophores was also taken into account. This implies that interconversion among conformational substates of probes linkages is slow on the time scale of the energy transfer process.     

Voltage sensitivity of the fluorescent probe RH421 in a model membrane system.

The voltage sensitivity of the fluorescent styrylpyridinium dye RH421 has been investigated in dimyristoylphosphatidylcholine vesicles by inducing an intramembrane electric field through the binding of the hydrophobic ion tetraphenylborate (TPB). To assess the probability of electrochromic and solvatochromic mechanisms for the dye response, the ground-state dipole moment of the dye in chloroform solution was determined from dielectric constant measurements to be 12 (+/- 1) Debye, and the change in dipole moment upon excitation was calculated from measurements of the Stokes shift in solvents of varying polarity to be 25 (+/- 11) Debye. As well as causing absorbance and fluorescence changes of membrane-bound dye, the TPB-induced electrical field was found to reduce significantly the pKa of the dye. The pH at which experiments are carried out is, thus, an important factor in determining the amplitude of the voltage-induced absorbance and fluorescence changes. The observed absorbance changes induced by the field are inconsistent with a pure electrochromic mechanism. A reorientation/solvatochromic mechanism, whereby the electrical field reorients the dye molecules so that they experience a change in polarity of their lipid environment is likely to make a significant contribution to both the spectral changes and to the field effect on the acid-base properties of the dye.     

Property of the M-DNA probed by a minor groove binding dye 4',6-diamidino-2-phenylindole

Spectral properties including circular and linear dichroism (CD and LD) of M-DNA, a molecular electric wire, formed at a high Zn(2+) concentration have been studied using a minor groove binding drug 4',6-diamidino-2-phenylindole (DAPI) as a probe. As the Zn(2+) concentration increased, the magnitude of LD in the DNA absorption region decreased at pH 8.5, implying the aggregation of DNA, which is in contrast with the retained LD magnitude at pH 7.0. As the M-DNA formed, change in the secondary structure of DNA was observed by CD spectrum, which resembles that of the C-form DNA, although overall structure seemed to remain as a right handed double helix. The DAPI-DNA complex in the presence of high concentration of Zn(2+) ions at pH 7.0 exhibited the similar CD spectrum with that in the absence of Zn(2+) ion, consisting of type I, II and III. In contrast, at pH 8.5 at a high Zn(2+) concentration in which DNA is in its M-form, DNA bound DAPI produced only the type III CD, suggesting that DAPI binds at the surface of M-DNA: the presence of Zn(2+) ions prevents the minor groove binding of DAPI.     

Near-infrared, dual-ratiometric fluorescent label for measurement of pH

We describe the spectral properties of an amine-reactive, pH-sensitive, long-wavelength ratiometric fluorescent label having a pK_a in the physiological pH range. The label exhibits its main absorption and emission in the near-infrared (NIR) region. On deprotonation, a blue shift of the excitation maximum is observed. Importantly, both the protonated and deprotonated forms of the label are fluorescent, with the deprotonated form having an extremely large Stokes shift of more than 100 nm. The spectral and photophysical properties of this pH label are compared with the properties of the protein-conjugated forms. Due to the observed pK_a shift to the acidic pH range upon conjugation to proteins, such labels are ideal for studying phagocytic events and their regulation by drugs and/or environmental factors.     

Long-range interactions between DNA-bound ligands

We have studied the interaction of the A:T specific minor-groove binding ligand 4′, 6-diamidino-2-phenylindole (DAPI) with synthetic DNA oligomers containing specific binding sites in order to investigate possible long-range interactions between bound ligands. We find that DAPI binds cooperatively to the oligomers. The degree of cooperativity increases with increasing number of binding sites and decreases with the separation between them. This dependence is paralleled by changes in the induced circular dichroism spectrum of DAPI, which decreases in intensity at 335 nm and increases at 365 nm. These results are consistent with an allosteric interaction of DAPI with DNA, where bound ligands cooperatively alter the structure of the DNA molecule. This structural change seems possible to induce under various conditions, including physiological. One consequence of allosteric binding is that ligands bound at a distance from each other sense each other's presence and influence each other's properties. If some regulatory proteins the same conformational change as DAPI, novel mechanisms for controlling gene expression can be anticipated.     

RNA targeting by DNA binding drugs: Structural,conformational and energetic aspects of the binding of quinacrine and DAPI to A-form and H-form of poly(rC)·poly(rG)

A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4′,6-diamidino-2-phenylindole (DAPI) with the right handed Watson–Crick base paired A-form and the left-handed Hoogsteen base paired H^L-form of poly(rC)·poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H^L-form of poly(rC)·poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H^L-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H^L-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H^L-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA–ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.     

Lysolecithin:lysolecithin acyltransferase from rabbit lung. A conformational study

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged. Addition of NaCl caused a progressive disordering of structure with a parallel increase of transacylase activity. The acid denaturation of the protein, at low and high ionic strengths, showed that the ionization of groups with pK in the range 5.9-6.4 was essential for denaturation. The structure was stable at basic pH. The addition of lipids resulted in a non-specific stabilization of the disordered conformation, in the same manner as the addition of NaCl. From these results, it is suggested that there are two conformations for this protein which differ in their ability to bind lysolecithin molecules in the enzyme deacylation step of the reaction. This hypothesis agrees with previously published properties of the enzyme, concerning aggregation with other proteins and kinetic data. From the amino acid composition and conformational properties, the authors suggest that this enzyme could be a peripheral membrane protein.     

Effect of sensor domain mutations on the properties of voltage-gated ion channels: molecular dynamics studies of the potassium channel Kv1.2

The effects on the structural and functional properties of the Kv1.2 voltage-gated ion channel, caused by selective mutation of voltage sensor domain residues, have been investigated using classical molecular dynamics simulations. Following experiments that have identified mutations of voltage-gated ion channels involved in state-dependent omega currents, we observe for both the open and closed conformations of the Kv1.2 that specific mutations of S4 gating-charge residues destabilize the electrostatic network between helices of the voltage sensor domain, resulting in the formation of hydrophilic pathways linking the intra- and extracellular media. When such mutant channels are subject to transmembrane potentials, they conduct cations via these so-called “omega pores.” This study provides therefore further insight into the molecular mechanisms that lead to omega currents, which have been linked to certain channelopathies.     

Green fluorescent protein ground states: the influence of a second protonation site near the chromophore

The photophysical properties of most green fluorescent protein mutants (GFPs) are strongly affected by pH. This effect must be carefully taken into account when using GFPs as fluorescent probes or indicators. Usually, the pH-dependence of GFPs is rationalized on the basis of the ionization equilibrium of the chromophore phenol group. Yet many different mutants show spectral behavior that cannot be explained by ionization of this group alone. In this study, we propose a general model of protonation comprising two ionization sites (2S model). Steady-state optical measurements at different pH and temperature and pH-jump relaxation experiments were combined to highlight the thermodynamic and kinetic properties of paradigmatically different GFP variants. Our experiments support the 2S model. For the case of mutants in which E222 is the second protonation site, thermodynamic coupling between this residue's and the chromophore's ionization reactions was demonstrated. In agreement with the 2S model predictions, X-ray analysis of one of these mutants showed the presence of two chromophore populations at high pH.     

Identification and Characterization of a Misfolded Monomeric Serpin Formed at Physiological Temperature

The native serpin state is kinetically trapped. However, under mildly destabilizing conditions, the conformational landscape changes, and a number of nonnative conformations with increased stability can be readily formed. The ability to undergo structural change is due to intrinsic strain within the serpin's tertiary fold, which is utilized for proteinase inhibition but renders the protein susceptible to aberrant folding and self-association. The relationship between these various conformations is poorly understood. Antichymotrypsin (ACT) is an inhibitory serpin that readily forms a number of inactive conformations, induced via either environmental stress or interaction with proteinases. Here we have used a variety of biophysical and structural techniques to characterize the relationship between some of these conformations. Incubation of ACT at physiological temperature results in the formation of a range of conformations, including both polymer and misfolded monomer. The ability to populate these nonnative states and the native conformation reflects an energy landscape that is very sensitive to the solution conditions. X-ray crystallography reveals that the misfolded monomeric conformation is in the delta conformation. Further polymerization and seeding experiments show that the delta conformation is an end point in the misfolding pathway of ACT and not an on-pathway intermediate formed during polymerization. The observation that ACT readily forms this inactive conformation at physiological temperature and pH suggests that it may have a role in both health and disease.