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1.
Felitsky DJ Cannon JG Capp MW Hong J Van Wynsberghe AW Anderson CF Record MT 《Biochemistry》2004,43(46):14732-14743
Paradoxically, glycine betaine (N,N,N-trimethyl glycine; GB) in vivo is both an effective osmoprotectant (efficient at increasing cytoplasmic osmolality and growth rate) and a compatible solute (without deleterious effects on biopolymer function, including stability and activity). For GB to be an effective osmoprotectant but not greatly affect biopolymer stability, we predict that it must interact very differently with folded protein surface than with that exposed in unfolding. To test this hypothesis, we quantify the preferential interaction of GB with the relatively uncharged surface exposed in unfolding the marginally stable lacI helix-turn-helix (HTH) DNA binding domain using circular dichroism and with the more highly charged surfaces of folded hen egg white lysozyme (HEWL) and bovine serum albumin (BSA) using all-gravimetric vapor pressure osmometry (VPO) and compare these results with results of VPO studies (Hong et al. (2004), Biochemistry, 43, 14744-14758) of the interaction of GB with polyanionic duplex DNA. For these four biopolymer surfaces, we observe that the extent of exclusion of GB per unit of biopolymer surface area increases strongly with increasing fraction of anionic oxygen (protein carboxylate or DNA phosphate) surface. In addition, GB is somewhat more excluded from the surface exposed in unfolding the lacI HTH and from the folded surface of HEWL than expected from their small fraction of anionic surface, consistent with moderate exclusion of GB from polar amide surface, as predicted by the osmophobic model of protein stability (Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963). Strong exclusion of GB from anionic surface explains how it can be both an effective osmoprotectant and a compatible solute; analysis of this exclusion yields a lower bound on the hydration of anionic protein carboxylate surface of two layers of water (>or=0.22 H(2)O A(-)(2)). 相似文献
2.
Two thermodynamic models have been developed to interpret the preferential accumulation or exclusion of solutes in the vicinity of biopolymer surface and the effects of these solutes on protein processes. The local-bulk partitioning model treats solute (and water) as partitioning between the region at/or near the protein surface (the local domain) and the bulk solution. The solvent exchange model analyzes a 1:1 competition between water and solute molecules for independent surface sites. Here we apply each of these models to interpret thermodynamic data for the interactions of urea and the osmoprotectant glycine betaine (N,N,N-trimethylglycine; GB) with the surface exposed in unfolding the marginally stable lacI HTH DNA binding domain. The partition coefficient K(P) quantifying accumulation of urea at this protein surface (K(P) approximately equal 1.1) is only weakly dependent on urea concentration up to 6 M urea. However, K(P) quantifying exclusion of GB from the vicinity of this protein surface increases from 0.83 (extrapolated to 0 M GB) to 1.0 (indicating that local and bulk GB concentrations are equal) at 4 M GB (activity > 40 M). We interpret the significant concentration dependence of K(P) for GB, predicted to be general for excluded, nonideal solutes such as GB, as a modest (8%) attenuation of the GB concentration dependence of solute nonideality in the local domain relative to that in the bulk solution. Above 4 M, K(P) for the interaction of GB with the surface exposed in protein unfolding is predicted to exceed unity, which explains the maximum in thermal stability observed for RNase and lysozyme at 4 M GB (Santoro, M. M., Liu, Y. F., Khan, S. M. A., Hou, L. X., and Bolen, D. W. (1992) Biochemistry 31, 5278-5283). Both thermodynamic models provide good two-parameter fits to GB and urea data for lacI HTH unfolding over a wide concentration range. The solute partitioning model allows for a full spectrum of attenuation effects in the local domain, encompasses the cases treated by the competitive binding model, and provides a somewhat better two-parameter fit of effects of high GB concentration on lacI HTH stability. Parameters of this fit should be applicable to isothermal and thermal unfolding data for all proteins with similar compositions of surface exposed in unfolding. 相似文献
3.
Thermodynamic analysis of urea-biopolymer interactions and effects of urea on folding of proteins and alpha-helical peptides shows that urea interacts primarily with polar amide surface. Urea is therefore predicted to be a quantitative probe of coupled folding, remodeling, and other large-scale changes in the amount of water-accessible polar amide surface in protein processes. A parallel analysis indicates that glycine betaine [N,N,N-trimethylglycine (GB)] can be used to detect burial or exposure of anionic (carboxylate, phosphate) biopolymer surface. To test these predictions, we have investigated the effects of these solutes (0-3 m) on the formation of 1:1 complexes between lac repressor (LacI) and its symmetric operator site (SymL) at a constant KCl molality. Urea reduces the binding constant K(TO) [initial slope dlnK(TO)/dm(urea) = -1.7 +/- 0.2], and GB increases K(TO) [initial slope dlnK(TO)/dm(GB) = 2.1 +/- 0.2]. For both solutes, this derivative decreases with an increase in solute concentration. Analysis of these initial slopes predicts that (1.5 +/- 0.3) x 10(3) A2 of polar amide surface and (4.5 +/- 1.0) x 10(2) A2 of anionic surface are buried in the association process. Analysis of published structural data, together with modeling of unfolded regions of free LacI as extended chains, indicates that 1.5 x 10(3) A2 of polar amide surface and 6.3 x 10(2) A2 of anionic surface are buried in complexation. Quantitative agreement between structural and thermodynamic results is obtained for amide surface (urea); for anionic surface (GB), the experimental value is approximately 70% of the structural value. For LacI-SymL binding, two-thirds of the structurally predicted change in amide surface (1.0 x 10(3) A2) occurs outside the protein-DNA interface in protein-protein interfaces formed by folding of the hinge helices and interactions of the DNA binding domain (DBD) with the core of the repressor. Since urea interacts principally with amide surface, it is particularly well-suited to detect and quantify the extent of coupled folding and other large-scale remodeling events in the steps of protein-nucleic acid interactions and other protein associations. 相似文献
4.
To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K(+)Glu(-)), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Gamma(mu1) and the corresponding dialysis preferential interaction coefficient Gamma(mu1),(mu3) (both calculated from the VPO data) are negative; Gamma(mu1), (mu3) is proportional to bulk solute molality (m(bulk)3) at least up to 1 m (molal). Negative values of Gamma(mu1),(mu3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Gamma(mu1),(mu3)/m(bulk)3 = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu1),(mu3)/m(bulk)3 = -10 +/- 1 m(-1)). Between these extremes, the magnitude of Gamma(mu1),(mu3)/m(bulk)3 decreases in the order glycine betaine > proline >TMAO > trehalose approximately K(+)Glu(-) > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Gamma(mu1),(mu3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H(2)O/BSA, which corresponds to slightly less than a monolayer (estimated to be approximately 3.2 x 10(3) H(2)O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account. 相似文献
5.
A simple molecular model for the thermodynamic behavior of non-polar solutes in water and in aqueous solutions of protein denaturants is presented. Three contributions are considered: (i) combinatorial arising from the mixing process, (ii) interactional characterizing the molecular interactions occurring in the mixture and (iii) a contribution originating from the structural changes occurring in the first shell of water molecules around the solute. The latter is modeled assuming that water molecules in contact with the solute are involved in a chemical equilibrium between two states. The model describes well the temperature and denaturant concentration dependences of the Gibbs energies of solution and transfer for benzene, toluene and alkanes in water and aqueous solutions of urea and guanidine hydrochloride. Model parameters are physically meaningful, allowing a discussion of the molecular interactions involved. A preferential solvation of the solute by the denaturant is found. However, the non-polar solute-denaturant interaction is not specific, i.e. leading to a distinct chemical entity. Urea and guanidine hydrochloride are non-polar solubilizing agents because their interactions with the solute are less unfavorable than those between water and the solute. 相似文献
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The melting temperature of the poly(dA) . poly(dT) double helix is exquisitely sensitive to salt concentration, and the helix-to-coil transition is sharp. Modern calorimetric instrumentation allows this transition to be detected and characterized with high precision at extremely low duplex concentrations. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influence of other solutes on solution properties. We demonstrate how the temperature associated with poly(dA) . poly(dT) melting can be used to define the change in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We use this information to critically evaluate features of counterion condensation theory, as well as to illustrate "crosstalk" between different, non-contacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA) . poly(dT), in the presence of excess genomic salmon sperm DNA, or in the presence of one of two synthetic RNA polymers (the poly(rA) . poly(rU) duplex or the poly(rU) . poly(rA) . poly(rU) triplex). We find that these additions cause a shift in the melting temperature of poly(dA) . poly(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed tm shift does not depend significantly on whether the added polymer is RNA or DNA, but it does depend on the number of strands making up the helix of the added polymer. We ascribe the observed changes in melting temperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and its associated counterions. We refer to this communication between non-contacting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of tm versus log[Na+] for poly(dA) . poly(dT), we estimate the magnitude of the apparent change in ionic strength resulting from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical considerations correctly predict the direction of the t(m) shift (the melting temperature increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5% of the concentration of the added duplex DNA or RNA (in mol phosphate), and an additional apparent increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent increase of 14.5 %. For the poly(rU) . poly(rA) . poly(rU) triplex, the total apparent increase in ionic strength corresponds to about 13.6% of the amount of added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation concentration induced by the presence and/or the transition of one of the solute molecules. We note that our results are general, so one can use a different solute probe sensitive to proton binding to characterize subtle changes in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probing counterion environments, and in terms of potential regulatory mechanisms. 相似文献
8.
Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data. 相似文献
9.
Investigation of nucleic acid interactions was performed using dual polarization interferometry, a novel approach to elucidating molecular interactions. This paper presents a preliminary study of adsorption of single stranded DNA onto functionalised silicon oxynitride, compared with covalent linkage, and avidin-biotin immobilisation. The effect of probe concentration on hybridisation efficiency was also examined. We found that increasing the electrolyte concentration resulted in a decrease of adsorbed DNA and that capture of a biotinylated duplex DNA on an adsorbed avidin layer resulted in four times fewer molecules per cm(2) than for duplex DNA covalently bound via an amine end terminal. The rate of thickness increase of a biotin probe layer on an adsorbed avidin capture layer increased 10-fold when the probe concentration was increased from 0.1 microM to 1 microM. The close grafting density of the higher concentration probe meant that the immobilised probes were unavailable for hybridisation. 相似文献
10.
The binding of the recA gene product from E. coli to double-stranded and single-stranded nucleic acids has been investigated by following the change in melting temperature of duplex DNA and the fluorescence of single-stranded DNA or poly(dA) modified by reaction with chloroacetaldehyde. At low ionic strength, in the absence of Mg2+ ions, RecA protein binds preferentially to duplex DNA or poly(dA-dT). This leads to an increase of the DNA melting temperature. Stabilization of duplex DNA decreases when ionic strength or pH increases. In the presence of Mg2+ ions, preferential binding to single-stranded polynucleotides is observed. Precipitation occurs when duplex DNA begins to melt in the presence of RecA protein. From competition experiments, different single-stranded and double-stranded polydeoxynucleotides can be ranked according to their ability to bind RecA protein. Structural changes induced in nucleic acids upon RecA binding are discussed together with conformational changes induced in RecA protein upon magnesium binding. 相似文献
11.
A quantitative characterization of the thermodynamic effects due to interactions of salt ions and urea in aqueous solution is needed for rigorous analyses of the effects of changing urea concentration on biopolymer processes in solutions that also contain salt. Therefore, we investigate preferential interactions in aqueous solutions containing KCl and urea by using vapor pressure osmometry (VPO) to measure osmolality as a function of the molality of urea (component 3) over the range 0.09相似文献
12.
PNA beacons for duplex DNA 总被引:12,自引:0,他引:12
Kuhn H Demidov VV Gildea BD Fiandaca MJ Coull JC Frank-Kamenetskii MD 《Antisense & nucleic acid drug development》2001,11(4):265-270
We report here on the hybridization of peptide nucleic acid (PNA)-based molecular beacons (MB) directly to duplex DNA sites locally exposed by PNA openers. Two stemless PNA beacons were tested, both featuring the same recognition sequence and fluorophore-quencher pair (Fluorescein and DABCYL, respectively) but differing in arrangement of these groups and net electrostatic charge. It was found that one PNA beacon rapidly hybridized, with the aid of openers, to its complementary target within duplex DNA at ambient conditions via formation of a PD-like loop. In contrast, the other PNA beacon bound more slowly to preopened duplex DNA target and only at elevated temperatures, although it readily hybridized to single-stranded (ss) DNA target. Besides a higher selectivity of hybridization provided by site-specific PNA openers, we expect this approach to be very useful in those MB applications when denaturation of the duplex DNA analytes is unfavorable or undesirable. Furthermore, we show that PNA beacons are advantageous over DNA beacons for analyzing unpurified/nondeproteinized DNA samples. This feature of PNA beacons and our innovative hybridization strategy may find applications in emerging fluorescent DNA diagnostics. 相似文献
13.
Unwinding of unnatural substrates by a DNA helicase 总被引:6,自引:0,他引:6
Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined. Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding. The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA). PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts. Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates. The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates. 相似文献
14.
Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces. 总被引:1,自引:0,他引:1
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M Fojta V Vetterl M Tomschik F Jelen P Nielsen J Wang E Palecek 《Biophysical journal》1997,72(5):2285-2293
Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions. 相似文献
15.
Belosludtsev Y Belosludtsev I Iverson B Lemeshko S Wiese R Hogan M Powdrill T 《Biochemical and biophysical research communications》2001,282(5):1263-1267
Hybridization rate enhancement has been demonstrated for high molecular weight DNA target binding to a microarray. Microarrays were fabricated using biotin-modified oligonucleotides complexed with streptavidin (SA), which serves as an attachment to the underlying surface. It is shown that at low salt and pH 5, where SA develops a positive charge, duplex formation becomes at least 80-fold faster than seen under standard conditions, where SA is neutral or anionic. Duplex formation becomes independent of solution state cation concentration in the low pH state, under conditions where specificity remains high. The utility of such applied surface science is discussed. 相似文献
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Deanpen Japrung 《Biophysical journal》2010,98(9):1856-1863
The staphylococcal α-hemolysin (αHL) protein nanopore is under investigation as a fast, cheap detector for nucleic acid analysis and sequencing. Although discrimination of all four bases of DNA by the αHL pore has been demonstrated, analysis of single-stranded DNAs and RNAs containing secondary structure mediated by basepairing is prevented because these nucleic acids cannot be translocated through the pore. Here, we show that a structured 95-nucleotide single-stranded DNA and its RNA equivalent are translocated through the αHL pore in the presence of 4 M urea, a concentration that denatures the secondary structure of the polynucleotides. The αHL pore is functional even in 7 M urea, and therefore it is easily stable enough for analyses of challenging DNA and RNA species. 相似文献
18.
G x A mismatched base pairs are frequently found in nucleic acids. Human centromere DNA sequences contain unusual repeating motifs, e.g. , (GAATG)n x (CATTC)n found in the human chromosome. The purine-rich strand of this repeating pentamer sequence forms duplex and hairpin structures with unusual stability. The high stability of these structures is contributed by the "sheared" G x A base pairs which present a novel recognition surface for ligands and proteins. We have solved the crystal structure, by the multiple-wavelength anomalous diffraction (MAD) method of d(CCGAATGAGG) in which the centromere core sequence motif GAATG is embedded. Three crystal forms were refined to near-atomic resolution. The structures reveal the detailed conformation of tandem G x A base pairs whose unique hydrogen-bonding surface has interesting interactions with bases, hydrated magnesium ions, cobalt(III)hexaammine, spermine, and water molecules. The results are relevant in understanding the structure associated with human centromere sequence in particular and G x A base pairs in nucleic acids (including RNA, like ribozyme) in general. 相似文献
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The human immunodeficiency virus type 1 nucleocapsid protein (NC) is a nucleic acid chaperone that catalyzes the rearrangement of nucleic acids into their thermodynamically most stable structures. In the present study, a combination of optical and thermodynamic techniques were used to characterize the influence of NC on the secondary structure, thermal stability and energetics of monomolecular DNA quadruplexes formed by the sequence d(GGTTGGTGTGGTTGG) in the presence of K+ or Sr2+. Circular dichroism studies demonstrate that NC effectively unfolds the quadruplexes. Studies carried out with NC variants suggest that destabilization is mediated by the zinc fingers of NC. Calorimetric studies reveal that NC destabilization is enthalpic in origin, probably owing to unstacking of the G-quartets upon protein binding. In contrast, parallel studies performed on a related DNA duplex reveal that under conditions where NC readily destabilizes and unfolds the quadruplexes, its effect on the DNA duplex is much less pronounced. The differences in NC's ability to destabilize quadruplex versus duplex is in accordance with the higher ΔG of melting for the latter, and with the inverse correlation between nucleic acid stability and the destabilizing activity of NC. 相似文献