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1.
Extent of binding (gamma 2(1)) of cationic surfactants cetyltrimethyl ammonium bromide (CTAB), myristyltrimethyl ammonium bromide (MTAB) and dodecyl trimethyl ammonium bromide (DTAB) to calf-thymus DNA, bovine serum albumin (BSA) and to their binary mixture respectively have been measured as function of bulk concentration of the surfactant by using equilibrium dialysis technique. Binding of CTAB has been studied at different pH, ionic strength (mu), temperature and biopolymer composition and with native and denatured states of the biopolymers. The chain-length of different long chain amines plays a significant role in the extent of binding under identical solution condition. The binding ratios for CTAB to collagen, gelatin, DNA-collagen and DNA-gelatin mixtures respectively have also been determined. The conformational structures of different biopolymers are observed to play significant role in macromolecular interactions between protein and DNA in the presence of CTAB. From the experimental values of the maximum binding ratio (gamma 2m) at the saturation level for each individual biopolymer, ideal values (gamma 2m)id have been theoretically calculated for binary mixtures of biopolymers using additivity rule. The protein-DNA-CTAB interaction in mixture has been explained in terms of the deviation (delta) of (gamma 2m) from (gamma 2m)id in the presence of a surfactant in bulk. The binding of surfactants to biopolymers and to their binary mixtures are compared more precisely in terms of the Gibbs' free energy decrease (-delta G degree) for the saturation of the binding sites in the biopolymers or biopolymer mixtures with the change of the bulk surfactant activity from zero to unity in the rational mole fraction scale. 相似文献
2.
Extent of binding (gammap) of globular proteins to calf-thymus DNA have been measured in mole per mole of nucleotide as function of equilibrium protein concentration. We have exploited measurement of the surface tension of the protein solution in the presence and absence of DNA to calculate the binding ration (gammap). Interaction of bovine serum albumin with DNA has been studied at different pH. Interaction of bovine serum albumin with DNA has been studied at different pH, ionic strength and in presence of Ca2+. Interaction of BSA with denatured DNA has also been investigated. Binding isotherms for other globular proteins like beta-lactoglobulin, alpha-lactalbumin and lysozyme have been compared under identical physicochemical condition. It has been noted with considerable interest that globular form of protein is important to some extent in protein-DNA interaction. An attempt has been made to explain the significance of difference in binding ratios of these two biopolymers in aqueous medium for different systems in the light of electrostatic and hydrophobic effects. Values of maximum binding ration (gammap(m)) at saturated level for different systems have been also presented. The Gibb's free energy decrease (-deltaG0) of the binding of proteins to DNA has been compared more precisely for the saturation of binding sites in the DNA with the change of activity of protein in solution from zero to unity in the rational mole fraction scale. 相似文献
3.
In an earlier publication by Chattoraj et al. [Biophysical Chemistry 63 (1996) 37], a generalized equation for standard free energy of (delta G0) interaction of surfactant, inorganic salts and aqueous solvent with protein, forming a single phase has been deduced on strict thermodynamic grounds. In the present paper, this equation has been utilized to calculate delta G0 in kilojoules per kilogram of different proteins for the change of bulk surfactant activity from zero to unity in the mole fraction scale. Values of binding interactions of CTAB, MTAB, DTAB and SDS to BSA, beta-lactoglobulin, gelatin, casein, myosin, lysozyme and their binary and ternary mixtures had already been determined in this laboratory at different surfactant concentrations, pH, ionic strength and temperature using an equilibrium dialysis technique. Values of delta G0 for saturated protein-surfactant complexes as well as unsaturated complexes are found to be equal. delta G0 is also found to vary linearly with maximum moles of surfactants bound to a kilogram of protein or protein mixture and the slope of this linear plot represents standard free energy delta G0B for the transfer of 1 mol of surfactant from the bulk for binding reaction with protein; -delta G0 values for different systems vary widely and the order of their magnitudes represents relative affinities of surfactants to proteins. Magnitude of -delta G0B on the other hand varies within a narrow range of 32-37 kJ/mol of surfactant. For interaction of SDS with BSA, close to the CMC, values of delta G0 are very high due to the formation of micelles of protein-bound surfactants. Values of delta G0 for negative binding of inorganic salts to proteins and protein mixtures have been evaluated using our generalized equation in which excess binding values of water and salts have been calculated from the data obtained from our previous isopiestic experiments. delta G0 values in these cases are positive due to the excess hydration of proteins. Negative values of delta G0 in surfactant interaction and positive values of delta G0 for hydration of proteins in the presence of neutral salts represent relative affinities of proteins for solute and solvent since in all cases, the reference state for delta G0 is the unit mole fraction of solute in the aqueous phase. 相似文献
4.
It is well established that the insertion of a nucleotide into a growing DNA chain requires a conformational change in the structure of a DNA polymerase. These enzymes have been shown to bind a primer-template in the open conformation and then upon binding of a complementary dNTP undergo a conformational rearrangement to the closed ternary complex. This movement results in the positioning of the incoming nucleotide in the proper geometry for the nucleophilic attack by the 3'-hydroxyl of the primer. In this work, tryptic digestion experiments were performed to detect this conformational change in the structure of the exonuclease-deficient DNA polymerase I (Klenow fragment). Three distinct digestion patterns were observed: one for the polymerase alone, one for the binary complex with the primer-template, and one for the ternary polymerase-DNA-dNTP complex. The latter conformational change leads to a stable ternary closed complex formation only when the correct nucleotide is present in the reaction mixture. Positioning of nucleotides with incorrect geometry in the protein active site inhibits or eliminates formation of the closed complex. Similarly, this conformational change is inhibited when the primer terminus of the DNA molecule is altered by the presence of the 2'-hydroxyl. 相似文献
5.
Sangita Kachhap Pragya Priyadarshini 《Journal of biomolecular structure & dynamics》2017,35(6):1153-1167
Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in Drosophila melanogaster are known to bind cooperatively to 5′-(T/C)TAATTAA(T/A)(T/A)G-3′ DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein–DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll–3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll–3A01–Al. In addition, this change in secondary structure of Cll does not allow it to form protein–protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al–3A01–Cll where Cll binds to Al–3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein–protein interactions while proposing likely mechanism of recognition in ternary complex, Al–Cll–DNA. 相似文献
6.
Liebert K Horton JR Chahar S Orwick M Cheng X Jeltsch A 《The Journal of biological chemistry》2007,282(31):22848-22855
The crystal structure of the Escherichia coli DNA adenine methyltransferase (EcoDam) in a binary complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) unexpectedly showed the bound AdoHcy in two alternative conformations, extended or folded. The extended conformation represents the catalytically competent conformation, identical to that of EcoDam-DNA-AdoHcy ternary complex. The folded conformation prevents catalysis, because the homocysteine moiety occupies the target Ade binding pocket. The largest difference between the binary and ternary structures is in the conformation of the N-terminal hexapeptide ((9)KWAGGK(14)). Cofactor binding leads to a strong change in the fluorescence of Trp(10), whose indole ring approaches the cofactor by 3.3A(.) Stopped-flow kinetics and AdoMet cross-linking studies indicate that the cofactor prefers binding to the enzyme after preincubation with DNA. In the presence of DNA, AdoMet binding is approximately 2-fold stronger than AdoHcy binding. In the binary complex the side chain of Lys(14) is disordered, whereas Lys(14) stabilizes the active site in the ternary complex. Fluorescence stopped-flow experiments indicate that Lys(14) is important for EcoDam binding of the extrahelical target base into the active site pocket. This suggests that the hexapeptide couples specific DNA binding (Lys(9)), AdoMet binding (Trp(10)), and insertion of the flipped target base into the active site pocket (Lys(14)). 相似文献
7.
Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor. 相似文献
8.
Characterization of partially folded intermediates of papain in presence of cationic, anionic, and nonionic detergents at low pH 总被引:1,自引:0,他引:1
A systematic investigation of the effects of detergents [Sodium dodecyl sulphate (SDS), hexa decyltrimethyl ammonium bromide (CTAB) and Tween-20] on the structure of acid-unfolded papain (EC.3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence, and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits a substantial amount of secondary structure and is relatively less denatured compared with 6 M GdnHCl (guanidine hydrochloride) but loses the persistent tertiary contacts of the native state. Addition of detergents caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Near-UV CD spectra also showed the regain of native-like spectral features in the presence of 8 mM SDS and 3.5 mM CTAB. Induction of structure in acid-unfolded papain was greater in the presence SDS followed by CTAB and Tween-20. Intrinsic tryptophan fluorescence studies indicate the change in the environment of tryptophan residues upon addition of detergents to acid-unfolded papain. Addition of 8 mM SDS resulted in the loss of ANS binding sites exhibited by a decrease in ANS fluorescence intensity, suggesting the burial of hydrophobic patches. Maximum ANS binding was obtained in the presence of 0.1 mM Tween-20 followed by CTAB, indicating a compact "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in the presence of detergents showed the partial recovery of enzymatic activity. These results suggest that papain at low pH and in the presence of SDS exists in a partially folded state characterized by native-like secondary structure and tertiary folds. While in the presence of Tween, acid-unfolded papain exists as a compact intermediate with molten-globule-like characteristics, viz. enhanced hydrophobic surface area and retention of secondary structure. While in the presence of CTAB it exists as a compact intermediate with regain of native-like secondary and partial tertiary structure as well as high ANS binding with the partially recovered enzymatic activity, i.e., a molten globule state with tertiary folds. 相似文献
9.
The binding of pancreatic ribonuclease-A by denatured DNA, native DNA, poly-dA, and poly-dT, has been studied by a gel filtration method. With denatured DNA at pH 7.5, ionic strength 0.053M, there is one binding site per 12 nucleotides and the equilibrium binding constant per site is 9.7 × 104 l./mole. The binding constant increases by a factor of 8 as the pH is decreased from 8 to 7. The strength of the binding of denatured DNA increases with decreasing ionic strength. At pH 7.5, native DNA binds about ? as strongly as does denatured DNA. The binding affinity increases in the order poly-dA, denatured DNA, and poly-dT. These results support the view that the binding of denatured DNA involves both electrostatic interactions between the negatively charged polynucleotide and the positively charged protein, and an interaction of the protein with a pyrimidine residue of the denatured DNA, and thus that the binding is basically similar to that between RNAse and its substrate RNA. 相似文献
10.
《Chemical Speciation and Bioavailability》2013,25(4):231-239
AbstractComplexation of toxic metal ions with maleic acid in (0.0–2.5% w/v) cetyltrimethylammonium bromide (CTAB)–water mixtures has been studied pH-metrically at ambient conditions and an ionic strength of 0.16 mol L-1. The existence of different binary species was established from modelling studies using the computer program MINIQUAD75. The best-fit chemical models were selected based on statistical parameters such as the crystallographic R factor and sum of the squares of residuals in mass-balance equations. The models for binary complex systems contain the chemical species ML2, ML2H and ML3 for Pb(II), Cd(II) and Hg(II) in CTAB–water mixtures. The trend in the variation of stability constants with change in the mole fraction of the medium was explained based on electrostatic and non-electrostatic forces. Distribution of the species with pH at different compositions of CTAB–water mixtures was also presented. 相似文献
11.
1H-NMR relaxation times are reported for native and thermally denatured lysozyme aqueous solutions measured as the function of the proton mole fraction in the sample. A two-exponential character of proton longitudinal relaxation function was observed for native lysozyme solutions: the fast component was attributed to the non-exchangeable protein protons, the slow one to water protons. Purely exponential decay of longitudinal magnetization was observed for the thermally denatured samples. This has been explained in terms of a fast spin exchange model. The contributions of the protein protons to the water proton relaxation rate in native and thermally denatured samples were determined, too. 相似文献
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15.
Fractional precipitation of plasmid DNA from lysate by CTAB 总被引:6,自引:0,他引:6
Lander RJ Winters MA Meacle FJ Buckland BC Lee AL 《Biotechnology and bioengineering》2002,79(7):776-784
Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA. 相似文献
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17.
Mapping of RNA polymerase binding sites in R12 derived plasmids carrying the replication-incompatibility region and the insertion element IS1. 总被引:4,自引:0,他引:4
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Interactions between Escherichia coli RNA polymerase holoenzyme and three small plasmid DNAs (pSM1, pSM2, and pSM15) derived from the drug resistant factor R12 have been studied. These plasmids carry the copy number and incompatibility determinants, the origin of DNA replication and the rep gene(s) necessary for plasmid replication. They also contain the insertion element IS1 and the putative finO cistron. Thirteen DNA segments within the largest of the three plasmids (pSM2) were able to form either a binary and/or ternary complex with RNA polymerase. A unique strong binding site was mapped within the left end of IS1. Five binding sites were found within the rep-cop-inc region. Four of these are weak binding sites whereas the fifth does not form a stable binary complex and was detected by ternary complex formation. A strong binding site was located in the putative finO region whereas the remaining six binding sites are located in regions with unidentified genetic functions. 相似文献
18.
Pitts SL Jablonksy MJ Duca M Dauzonne D Monneret C Arimondo PB Anklin C Graves DE Osheroff N 《Biochemistry》2011,50(22):5058-5066
Etoposide is a widely prescribed anticancer drug that stabilizes covalent topoisomerase II-cleaved DNA complexes. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. Interactions between human topoisomerase IIα and etoposide in the binary enzyme--drug complex appear to be mediated by substituents on the A-, B-, and E-rings of etoposide. These protein--drug contacts in the binary complex have predictive value for the actions of etoposide within the ternary topoisomerase IIα--drug--DNA complex. Although the D-ring of etoposide does not appear to contact topoisomerase IIα in the binary complex, etoposide derivatives with modified D-rings display reduced cytotoxicity against murine leukemia cells [Meresse, P., et al. (2003) Bioorg. Med. Chem. Lett. 13, 4107]. This finding suggests that alterations in the D-ring may affect etoposide activity toward topoisomerase IIα in the ternary enzyme--drug--DNA complex. Therefore, to address the potential contributions of the D-ring to the activity of etoposide, we characterized drug derivatives in which the C13 carbonyl was moved to the C11 position (retroetoposide and retroDEPT) or the D-ring was opened (D-ring diol). All of the D-ring alterations decreased the ability of etoposide to enhance DNA cleavage mediated by human topoisomerase IIα in vitro and in cultured cells. They also weakened etoposide binding in the ternary enzyme--drug--DNA complex and altered sites of enzyme-mediated DNA cleavage. On the basis of these findings, we propose that the D-ring of etoposide has important interactions with DNA in the ternary topoisomerase II cleavage complex. 相似文献
19.
Nagata T Takada Y Ono A Nagata K Konishi Y Nukina T Ono M Matsugami A Furukawa A Fujimoto N Fukuda H Nakagama H Katahira M 《Nucleic acids research》2008,36(21):6816-6824
We found that UP1, a proteolytic product of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), both enhances and represses the telomerase activity. The formation of the UP1–telomerase RNA–telomeric DNA ternary complex was revealed by a gel retardation experiment. The interactions in the ternary and binary complexes were elucidated by NMR. UP1 has two nucleic acid-binding domains, BD1 and BD2. In the UP1–telomerase RNA binary complex, both BD1 and BD2 interact with telomerase RNA. Interestingly, when telomeric DNA was added to the binary complex, telomeric DNA bound to BD1 in place of telomerase RNA. Thus, BD1 basically binds to telomeric DNA, while BD2 mainly binds to telomerase RNA, which resulted in the formation of the ternary complex. Here, UP1 bridges telomerase and telomeric DNA. It is supposed that UP1/hnRNP A1 serves to recruit telomerase to telomeric DNA through the formation of the ternary complex. A model has been proposed for how hnRNP A1/UP1 contributes to enhancement of the telomerase activity through recruitment and unfolding of the quadruplex of telomeric DNA. 相似文献
20.
Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.
Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase. 相似文献