Separation-based approach to study dissociation kinetics of noncovalent DNA-multiple protein complexes

Noncovalent binding of DNA with multiple proteins is pivotal to many regulatory cellular processes. Due to the lack of experimental approaches, the kinetics of assembly and disassembly of DNA-multiple proteins complexes have never been studied. Here, we report on a first method capable of measuring disassembly kinetics of such complexes. The method is based on continuous spatial separation of different complexes. The kinetics of multiple complex dissociation processes are also spatially separated, which in turn facilitates finding their rate constants. Our separation-based approach was compared with a conventional no-separation approach by using computer simulation of dissociation kinetics. It proved to be much more accurate than the no-separation approach and to be a powerful tool for testing hypothetical mechanisms of the disassembly of DNA-multiple proteins complexes. An experimental implementation of the separation-based approach was finally demonstrated by using capillary electrophoresis as a separation method. The interaction between an 80 nucleotide long single-stranded DNA and single-stranded DNA binding protein was studied. DNA-protein complexes with one and two proteins were observed, and rate constants of their dissociation were determined. We foresee that a separation approach will be also developed to study the kinetics of the formation of DNA-multiple protein complexes.     

Exploiting Complex Fluorophore Interactions to Monitor Virus Capsid Disassembly

Supramolecular protein complexes are the corner stone of biological processes; they are essential for many biological functions. Unraveling the interactions responsible for the (dis)assembly of these complexes is required to understand nature and to exploit such systems in future applications. Virus capsids are well-defined assemblies of hundreds of proteins and form the outer shell of non-enveloped viruses. Due to their potential as a drug carriers or nano-reactors and the need for virus inactivation strategies, assessing the intactness of virus capsids is of great interest. Current methods to evaluate the (dis)assembly of these protein assemblies are experimentally demanding in terms of instrumentation, expertise and time. Here we investigate a new strategy to monitor the disassembly of fluorescently labeled virus capsids. To monitor surfactant-induced capsid disassembly, we exploit the complex photophysical interplay between multiple fluorophores conjugated to capsid proteins. The disassembly of the capsid changes the photophysical interactions between the fluorophores, and this can be spectrally monitored. The presented data show that this low complexity method can be used to study and monitor the disassembly of supramolecular protein complexes like virus capsids. However, the range of labeling densities that is suitable for this assay is surprisingly narrow.     

A new mathematical treatment of spectrophotometric data for differentiating mononuclear and polynuclear complexes

A new mathematical approach to the treatment of spectrophotometric data for differentiation between mononuclear and polynuclear metal complexes in solution is presented. The slope of a straight line yields the total number of units in the complex, from which the true molecular formula can be obtained if the empirical formula is known. The formation constant is also given by the slope of a straight line. The method can be applied to complexes with any number of different ligands. Solutions are prepared in which the ratio of total concentrations of the reacting species is equal to the combining ratio. Spectrophotometric data must be obtained under conditions where only one complex is formed and the degree of dissociation is significant. The molar absorptivity must be obtained by direct measurement.     

Thermal activation of the co-chaperonins GroES and gp31 probed by mass spectrometry

Many biological active proteins are assembled in protein complexes. Understanding the (dis)assembly of such complexes is therefore of major interest. Here we use mass spectrometry to monitor the disassembly induced by thermal activation of the heptameric co-chaperonins GroES and gp31. We use native electrospray ionization mass spectrometry (ESI-MS) on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to monitor the stoichiometry of the chaperonins. A thermally controlled electrospray setup was employed to analyze conformational and stoichiometric changes of the chaperonins at varying temperature. The native ESI-MS data agreed well with data obtained from fluorescence spectroscopy as the measured thermal dissociation temperatures of the complexes were in good agreement. Furthermore, we observed that thermal denaturing of GroES and gp31 proceeds via intermediate steps of all oligomeric forms, with no evidence of a transiently stable unfolded heptamer. We also evaluated the thermal dissociation of the chaperonins in the gas phase using infrared multiphoton dissociation (IRMPD) for thermal activation. Using gas-phase activation the smaller (2-4) oligomers were not detected, only down to the pentamer, whereafter the complex seemed to dissociate completely. These results demonstrate clearly that conformational changes of GroES and gp31 due to heating in solution and in the gas phase are significantly different.     

Kinetics and mechanism of iron(III) dissociation from the dihydroxamate siderophores alcaligin and rhodotorulic acid

The kinetics and mechanism of siderophore ligand dissociation from their fully chelated Fe(III) complexes is described for the highly preorganized cyclic tetradentate alcaligin and random linear tetradentate rhodotorulic acid in aqueous solution at 25 degrees C (Fe2L3 + 6H+ reversible 2 Fe3+ aq + 3 H2L). At siderophore:Fe(III) ratios where Fe(III) is hexacoordinated, kinetic data for the H(+)-driven ligand dissociation from the Fe2L3 species is consistent with a singly ligand bridged structure for both the alcaligin and rhodotorulic acid complexes. Proton-driven ligand dissociation is found to proceed via parallel reaction paths for rhodotorulic acid, in contrast with the single path previously observed for the linear trihydroxamate siderophore ferrioxamine B. Parallel paths are also available for ligand dissociation from Fe2(alcaligin)3, although the efficiency of one path is greatly diminished and dissociation of the bis coordinated complex Fe(alcaligin)(OH2)2+ is extremely slow (k = 10(-5) M-1 s-1) due to the high degree of preorganization in the alcaligin siderophore. Mechanistic interpretations were further confirmed by investigating the kinetics of ligand dissociation from the ternary complexes Fe(alcaligin)(L) in aqueous acid where L = N-methylacetohydroxamic acid and glycine hydroxamic acid. The existence of multiple ligand dissociation paths is discussed in the context of siderophore mediated microbial iron transport.     

Direct observation of enhanced translocation of a homeodomain between DNA cognate sites by NMR exchange spectroscopy

A novel approach is presented for studying the kinetics of specific protein-DNA interactions by NMR exchange spectroscopy. The experimental design involves the direct observation of translocation of a homeodomain between cognate sites on two oligonucleotide duplexes, differing by only a single base pair at the edge of the DNA recognition sequence. The single base-pair change perturbs the 1H-15N correlation spectrum of a number of residues, while leaving the affinity for the DNA unchanged. The exchange process has apparent rate constants in the 5-20 s-1 range which are linearly dependent upon the concentration of free DNA. These rates are about 3 orders of magnitude larger than the dissociation rate constant determined by gel shift assays at nanomolar DNA concentrations. The complete NMR exchange data set, comprising auto- and cross-peak intensities as a function of mixing time at five concentrations of free DNA, can be fit simultaneously to a simple model in which protein translocation between DNA duplexes occurs via a second-order process (with rate constants of approximately 6 x 104 M-1 s-1) involving direct collision of a protein-DNA complex with free DNA. This is akin to intersegmental transfer, and a physical model for the process is discussed. Rapid translocation at high concentrations of free DNA observed directly by NMR exchange spectroscopy reconciles the long half-lives of protein-DNA complexes measured by biochemical analysis in vitro with the highly dynamic behavior of such complexes observed in vivo. The relevance of this mechanism to the kinetics of protein-DNA interactions within the cell is discussed.     

Mechanism and kinetics of ligand exchange between ferric citrate and desferrioxamine B

The kinetics of ligand exchange between ferric citrate and desferrioxamine B (DFB) was investigated at pH 8.0 and high citrate/Fe molar ratios (500-5000) with particular attention given to understanding the precise mechanism of ligand exchange. Ferric citrate complexes present in a test solution and therefore involved in the reaction with the incoming ligand (DFB) were initially examined by evaluating ferric citrate speciation on the basis of published thermodynamic constants. The speciation analysis indicated that mononuclear (mono- and dicitrate) ferric complexes are the major species responsible for the ligand exchange with DFB under the conditions examined in the present work. Given the tendency of DFB to adjunctively associate with the ferric citrate complexes, we propose a kinetic model containing the following three mechanisms: (i) direct association of DFB to the ferric dicitrate complex prior to any dissociation of citrate molecules from the Fe center, (ii) adjunctive association of DFB toward ferric monocitrate complex following dissociation of one molecule of citrate from the parent complex, and (iii) complexation of hydrated Fe by DFB after sequential dissociation of two molecules of citrate from the Fe center. Overall rates for the ligand exchange were determined by spectrophotometrically monitoring the formation of ferrioxamine B. Further analysis in quantifying the rate of each mechanism by use of published and determined rate constants of relevant elemental reactions suggested that the first and second mechanisms were significant under our experimental conditions where [Cit] ? [DFB] with the relative importance of these two pathways depending on citrate concentration.     

Protolytic dissociation mechanisms and comparative acid stabilities of palladium(II), zinc(II), copper(II), and nickel(II) complexes of alkylated dipyrrins

Complexes of Pd(II), Cu(II), Ni(II), and Zn(II) with alkylated dipyrrins (Hdpm) were synthesized and characterized by physicochemical and spectroscopic methods. Protolytic dissociation kinetics of these complexes in benzene in the presence of acetic and trichloroacetic acid was studied. A protonated dipyrrin is the reaction product of protolytic dissociation of the complexes in acid solutions. The observed and true dissociation rate constants, as well as activation reaction parameters, were calculated. Kinetic models of the processes are proposed, and the patterns of influence of the ligand nature on dissociation kinetics were determined. The Pd(II) complexes proved to be much more stable than other those of the other metals, according to the results of the kinetic studies. The lability of the complexes strongly depends on the length and position of the alkyl substituent of the ligand. The dissociation of the Ni(II) complex gives a heteroligand complex at low concentrations of acid, but the complex undergoes full protolytic dissociation at higher concentrations of acid. The dissociation of the complex of Cu(II) is an equilibrium process, involving formation of the protonated form of the ligand.     

A method for analysis of multicomponent systems of interacting aromatic molecules in solution

A method has been developed for analyzing multicomponent mixtures containing N different types of noncovalently interacting aromatic molecules using spectroscopic data [nuclear magnetic resonance (NMR), UV-vis, fluorescence, circular dichroism]. The method is based on an algorithmical approach to modeling of the N-component dynamic equilibrium (N-STOCH algorithm), dealing with numbers in N-based numerical systems as analogs of molecular complexes being formed in solution. A basic property of the algorithm is the ability to incorporate any known specificity of molecular interactions without constructing complex mathematical formulas. The utility of the N-STOCH algorithm was demonstrated by using as an example the NMR investigation of the dissociation of a heteroassociation complex of two anticancer drugs on addition of caffeine.     

Interactions of ginsenosides with DNA duplexes： A study by electrospray ionization mass spectrometry and UV absorption spectroscopy

The non-covalent complexes of duplexes DNA and 9 ginsenosides（1 aglycone and 8 glycosides） were investigated using electrospray ionization mass spectrometry（ESI-MS） in the gas phase. The results of relative binding affinities in negative ion mode revealed that several factors impact on the duplexbinding properties of ginsenosides. Glycosylations of 20（S）-protopanaxadiol ginsenosides at the position C-20 and 20（S）-protopanaxatriol classification at the position C-6 enhanced the fraction of bound DNA sharply. A rhamnose moiety shows little lower binding intensities than glucose at the same position.Ginsenosides of 20（S）-protopanaxatriol result in subtle higher binding affinities toward the duplex DNA than 20（S）-protopanaxadiol family. However, glycosylation with two sugar moieties does not show a higher binding affinity than with only one moiety. The collision-induced dissociation experimental data demonstrate the gas-phase stability and fragmentation patterns of the ginsenoside/DNA complexes are related to the glycoside number. Positive ion ESI mass spectra of the complexes were also recorded. The result of ESI-MS suggests that hydrogen bonds are the dominate interaction between ginsenosides and DNA. Similar results were obtained in solution-phase by UV spectroscopy, which exhibit a hyperchromism and blue-shift effect when DNA solution was titrated by individual ginsenoside.     

Evaluation of binding affinity of protein-mutant dna complexes in solution by laser spray mass spectrometry

We have applied laser spray mass spectrometry developed by Hiraoka et al. to investigate the binding affinity of protein-mutant DNA complexes. The results were compared with our previous data of collision-induced dissociation (CID) experiments using electrospray ionization mass spectrometry (ESI-MS). Systematic experiments were carried out on the complexes of the c-Myb DNA binding domain (c-Myb DBD) bound to eight kinds of 16- or 22-mer point mutant double-stranded DNA (dsDNA), whose solution K(d) values are different in the range from 10(-9) M to 10(-7) M. The dissociation curve as a function of laser power was plotted for each complex, and the laser power where 50% of complex was dissociated (E(50%)) in population was obtained. The correlation coefficient between E(50%) and the relative binding free-energy change (DeltaDeltaG) of each complex formation in solutions was 0.9808, which is much better than the coefficient obtained by the previous ESI-CID experiments that was 0.859. In addition, complexes of the c-Myb DBD with five other mutant dsDNA were also examined to confirm that laser spray can be used to estimate the K(d) values of a DNA-protein complex in solutions if an appropriate calibration curve is available. In the process of laser spray, dissociations of these noncovalent complexes occur in solutions, but not in the gas phase. This differs greatly from ESI-CID. Laser spray mass spectrometry has been found to be better than ESI-CID in evaluating binding affinity of a protein to various mutant DNA.     

Gallium(III) complexes of DOTA and DOTA-monoamide: kinetic and thermodynamic studies

Given the practical advantages of the (68)Ga isotope in positron emission tomography applications, gallium complexes are gaining increasing importance in biomedical imaging. However, the strong tendency of Ga(3+) to hydrolyze and the slow formation and very high stability of macrocyclic complexes altogether render Ga(3+) coordination chemistry difficult and explain why stability and kinetic data on Ga(3+) complexes are rather scarce. Here we report solution and solid-state studies of Ga(3+) complexes formed with the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, (DOTA)(4-), and its mono(n-butylamide) derivative, (DO3AM(Bu))(3-). Thermodynamic stability constants, log K(GaDOTA) = 26.05 and log K(GaDO3AM(Bu)) = 24.64, were determined by out-of-cell pH-potentiometric titrations. Due to the very slow formation and dissociation of the complexes, equilibration times of up to ～4 weeks were necessary. The kinetics of complex dissociation were followed by (71)Ga NMR under both acidic and alkaline conditions. The GaDOTA complex is significantly more inert (τ(1/2) ～12.2 d at pH = 0 and τ(1/2) ～6.2 h at pH = 10) than the GaDO3AM(Bu) analogue (τ(1/2) ～2.7 d at pH = 0 and τ(1/2) ～0.7 h at pH = 10). Nevertheless, the kinetic inertness of both chelates is extremely high and approves the application of Ga(3+) complexes of such DOTA-like ligands in molecular imaging. The solid-state structure of the GaDOTA complex, crystallized from a strongly acidic solution (pH < 1), evidenced a diprotonated form with protons localized on the free carboxylate pendants.     

NMR distinction of single- and multiple-mode binding of small-molecule protein ligands

Identifying and characterizing small-molecule inhibitors of protein-protein interactions is of high interest for drug discovery and for chemical genetics studies of biological pathways. Very often, initial hits or first-generation compounds have low micromolar dissociation constants and cause line broadening in NMR spectra. It is very important for subsequent structure-based compound optimization to know if this line broadening is caused by intermediate exchange of the dissociation kinetics only or in addition by multiple binding modes. Here, we present an approach of how to distinguish these two situations and demonstrate its experimental application. Two very similar small-molecule ligands of Bcl-xL are considered that cause both severe line broadening of interface residues. We show that one compound exhibits single-mode binding, and broadening is just due to dissociation kinetics in the intermediate exchange regime, and the line broadening can be overcome by providing excess ligand. In the other case, line broadening is due to dissociation kinetics and exchange between multiple bound conformations, and broadening cannot be overcome by providing excess ligand. The procedures used are very general and can also be applied to characterizing protein-protein and protein-nucleic acid interactions.     

Energetics and dynamics of electron transfer and proton transfer in dissociation of metal(III)(salen)-peptide complexes in the gas phase

Time- and collision energy-resolved surface-induced dissociation (SID) of ternary complexes of Co(III)(salen)+, Fe(III)(salen)+, and Mn(III)(salen)+ with several angiotensin peptide analogues was studied using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Time-resolved fragmentation efficiency curves (TFECs) were modeled using an RRKM-based approach developed in our laboratory. The approach utilizes a very flexible analytical expression for the internal energy deposition function that is capable of reproducing both single-collision and multiple-collision activation in the gas phase and excitation by collisions with a surface. The energetics and dynamics of competing dissociation pathways obtained from the modeling provides important insight on the competition between proton transfer, electron transfer, loss of neutral peptide ligand, and other processes that determine gas-phase fragmentation of these model systems. Similar fragmentation behavior was obtained for various Co(III)(salen)-peptide systems of different angiotensin analogues. In contrast, dissociation pathways and relative stabilities of the complexes changed dramatically when cobalt was replaced with trivalent iron or manganese. We demonstrate that the electron-transfer efficiency is correlated with redox properties of the metal(III)(salen) complexes (Co > Fe > Mn), while differences in the types of fragments formed from the complexes reflect differences in the modes of binding between the metal-salen complex and the peptide ligand. RRKM modeling of time- and collision-energy-resolved SID data suggests that the competition between proton transfer and electron transfer during dissociation of Co(III)(salen)-peptide complexes is mainly determined by differences in entropy effects while the energetics of these two pathways are very similar.     

Separation of single‐base sequential isomers of single‐stranded DNA by capillary electrophoresis and its application in the discrimination of single‐base DNA mutations

Separation of single‐base substitution sequential DNA isomers remains one of the most challenging tasks in DNA separation by capillary electrophoresis. We developed a simple, versatile capillary electrophoresis technique for the separation of single‐base sequential isomers of DNA having the same chain length. This technique is based on charge differences resulting from the different protonation (acid dissociation) properties of the four DNA bases. A mixture of 13 single‐base sequential isomers of 12‐mer single‐stranded DNA was separated by using an electrophoretic buffer solution containing 20 mM phosphoric acid (pH 2.0) and 8 M urea. We demonstrated that our method could separate all possible mutation patterns under identical experimental conditions. In addition, application of our method to the separation of the polymerase chain reaction product of a 68‐mer gene fragment and its single‐base isomers indicates that in combination with the appropriate genomic DNA extraction techniques, the method can detect single‐base gene mutations.     

Selective dissociation of non-covalent bonds in biological molecules by laser spray

The laser spray developed in our laboratory was applied to the analysis of bovine serum albumin (BSA), double-stranded DNA (dsDNA) and a protein-DNA complex. The tip of a stainless-steel capillary was irradiated with a 10.6 micro m infrared laser by increasing the laser power from 0 W (electrospray) to 1.4 W. The laser beam was focused to about 0.3 mm at the tip of the stainless-steel capillary. When BSA aqueous solution was irradiated by the laser, highly charged monomer ions were newly observed in addition to the multiply charged ions of non-denatured monomer, dimer and trimer moieties. This indicates that BSA suffers from denaturation on irradiation with an infrared laser in solution. A 1.4 W laser power is not sufficient to cause the complete denaturation of BSA under the present experimental conditions. Whereas dsDNA was found to dissociate almost completely to single-stranded DNA constituents on laser irradiation with a power of 1.2 W, no fragmentation of DNA molecules was observed. For a protein-DNA complex, i.e. a complex of c-Myb DNA binding domain and dsDNA, dissociation of the complex to the component moieties was observed. These findings indicate that the laser spray can selectively dissociate non-covalent complexes into subunits without causing dissociation of the covalent bonds of the subunits. The laser spray will be a versatile method for the investigation of the structures and stabilities of biomolecules including non-covalent complexes.     

Bridging Ligand Length Controls AT Selectivity and Enantioselectivity of Binuclear Ruthenium Threading Intercalators

The slow dissociation of DNA threading intercalators makes them interesting as model compounds in the search for new DNA targeting drugs, as there appears to be a correlation between slow dissociation and biological activity. Thus, it would be of great value to understand the mechanisms controlling threading intercalation, and for this purpose we have investigated how the length of the bridging ligand of binuclear ruthenium threading intercalators affects their DNA binding properties. We have synthesised a new binuclear ruthenium threading intercalator with slower dissociation kinetics from ct‐DNA than has ever been observed for any ruthenium complex with any type of DNA, a property that we attribute to the increased distance between the ruthenium centres of the new complex. By comparison with previously studied ruthenium complexes, we further conclude that elongation of the bridging ligand reduces the sensitivity of the threading interaction to DNA flexibility, resulting in a decreased AT selectivity for the new complex. We also find that the length of the bridging ligand affects the enantioselectivity with increasing preference for the ΔΔ enantiomer as the bridging ligand becomes longer.     

Impact of ligand protonation on higher-order metal complexation kinetics in aqueous systems

The impact of ligand protonation on the complexation kinetics of higher-order complexes is quantitatively described. The theory is formulated on the basis of the usual situation for metal complex formation in aqueous systems in which the exchange of water for the ligand in the inner coordination sphere is rate-determining (Eigen mechanism). We derive expressions for the general case of lability of ML(n) species that account for the contributions from all outer-sphere complexes to the rate of complex formation. For dynamic complexes, dissociation of ML is usually the rate-determining step in the overall process ML(n) --> M. Under such conditions, it is the role of ligand protonation in the step ML --> M that is relevant for the kinetic flux. 1:2 complexes of Cd(II) with pyridine-2,6-dicarboxylic acid fall into this category, and their lability at a microelectrode is reasonably well predicted by the differentiated approach. For non-dynamic systems, the kinetic flux arising from dissociation of higher-order complexes contributes to the rate-determining step. In this case, the weighted contribution of protonated and unprotonated outer-sphere complexes in all contributing dissociation reactions must be taken into account. The kinetic flux arising from the dissociation of 1:2 complexes of Ni(II) with bicine at a conventional electrode was quite well described by this combined approach. The results establish the generic role of ligand protonation within the overall framework of metal complexation kinetics in which complexes may be dynamic to an extent that depends on the operational time scale of the measurement technique.