The Effect of Small Cosolutes that Mimic Molecular Crowding Conditions on the Stability of Triplexes Involving Duplex DNA.
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ABSTRACT:
SUBMITTER: Avino A
PROVIDER: S-EPMC4783943 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The intracellular environment is crowded and heterogeneous. Although the thermodynamic stability of nucleic acid duplexes is predictable in dilute solutions, methods of predicting such stability under specific intracellular conditions are not yet available. We recently showed that the nearest-neighbor model for self-complementary DNA is valid under molecular crowding condition of 40% polyethylene glycol with an average molecular weight of 200 (PEG 200) in 100 mM NaCl. Here, we determined nearest-neighbor parameters for DNA duplex formation under the same crowding condition to predict the thermodynamics of DNA duplexes in the intracellular environment. Preferential hydration of the nucleotides was found to be the key factor for nearest-neighbor parameters in the crowding condition. The determined parameters were shown to predict the thermodynamic parameters (∆H°, ∆S°, and ∆G°37) and melting temperatures (T m) of the DNA duplexes in the crowding condition with significant accuracy. Moreover, we proposed a general method for predicting the stability of short DNA duplexes in different cosolutes based on the relationship between duplex stability and the water activity of the cosolute solution. The method described herein would be valuable for investigating biological processes that occur under specific intracellular crowded conditions and for the application of DNA-based biotechnologies in crowded environments.
| S-EPMC7321957 | biostudies-literature
Project description:Long accepted as the most important interaction, recent work shows that steric repulsions alone cannot explain the effects of macromolecular cosolutes on the equilibrium thermodynamics of protein stability. Instead, chemical interactions have been shown to modulate, and even dominate, crowding-induced steric repulsions. Here, we use 19F NMR to examine the effects of small and large cosolutes on the kinetics of protein folding and unfolding using the metastable 7 kDa N-terminal SH3 domain of the Drosophila signaling protein drk (SH3), which folds by a two-state mechanism. The small cosolutes consist of trimethylamine N-oxide and sucrose, which increase equilibrium protein stability, and urea, which destabilizes proteins. The macromolecules comprise the stabilizing sucrose polymer, Ficoll, and the destabilizing globular protein, lysozyme. We assessed the effects of these cosolutes on the differences in free energy between the folded state and the transition state and between the unfolded ensemble and the transition state. We then examined the temperature dependence to assess changes in activation enthalpy and entropy. The enthalpically mediated effects are more complicated than suggested by equilibrium measurements. We also observed enthalpic effects with the supposedly inert sucrose polymer, Ficoll, that arise from its macromolecular nature. Assessment of activation entropies shows important contributions from solvent and cosolute, in addition to the configurational entropy of the protein that, again, cannot be gleaned from equilibrium data. Comparing the effects of Ficoll to those of the more physiologically relevant cosolute lysozyme reveals that synthetic polymers are not appropriate models for understanding the kinetics of protein folding in cells.
| S-EPMC5982521 | biostudies-literature
Project description:RNA performs various spatiotemporal functions in living cells. As the solution environments significantly affect the stability of RNA duplexes, a stability prediction of the RNA duplexes in diverse crowded conditions is required to understand and modulate gene expression in heterogeneously crowded intracellular conditions. Herein, we determined the nearest-neighbor (NN) parameters for RNA duplex formation when subjected to crowding conditions with an ionic concentration relevant to that found in cells. Determination of the individual contributions of excluded volume effect and water activity to each of the NN parameters in crowded environments enabled prediction of the thermodynamic parameters and their melting temperatures for plenty of tested RNA duplex formation in vitro and in cell with significant accuracy. The parameters reported herein will help predicting RNA duplex stability in different crowded environments, which will lead to an improved understanding of the stability-function relationship for RNAs in various cellular organelles with different molecular environments.
| S-EPMC10201447 | biostudies-literature
Project description:Protein enzymes are the main catalysts in the crowded and complex cellular interior, but their activity is almost always studied in dilute buffered solutions. Studies that attempt to recreate the cellular interior in vitro often utilize synthetic polymers as crowding agents. Here, we report the effects of the synthetic polymer cosolutes Ficoll, dextran, and polyvinylpyrrolidone, and their respective monomers, sucrose, glucose, and 1-ethyl-2-pyrrolidone, on the activity of the 18-kDa monomeric enzyme, Escherichia coli dihydrofolate reductase. At low concentrations, reductase activity increases relative to buffer and monomers, suggesting a macromolecular effect. However, the effect decreases at higher concentrations, approaching, and, in some cases, falling below buffer values. We also assessed activity in terms of volume occupancy, viscosity, and the overlap concentration (where polymers form an interwoven mesh). The trends vary with polymer family, but changes in activity are within threefold of buffer values. We also compiled and analyzed results from previous studies and conclude that alterations of steady-state enzyme kinetics in solutions crowded with synthetic polymers are idiosyncratic with respect to the crowding agent and enzyme.
| S-EPMC5699487 | biostudies-literature
Project description:A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion dependence of the thermodynamic stability for DNA triplexes. We focus on two experimentally studied systems: a 24-base DNA triplex and a pair of interacting 14-base triplexes. Our theoretical calculations for the number of bound ions indicate that the TBI model provides improved predictions for the number of bound ions than the classical Poisson-Boltzmann (PB) equation. The improvement is more significant for a triplex, which has a higher charge density than a duplex. This is possibly due to the higher ion concentration around the triplex and hence a stronger ion correlation effect for a triplex. In addition, our analysis for the free energy landscape for a pair of 14-mer triplexes immersed in an ionic solution shows that divalent ions could induce an attractive force between the triplexes. Furthermore, we investigate how the protonated cytosines in the triplexes affect the stability of the triplex helices.
| S-EPMC3427753 | biostudies-literature
Project description:Examining the effects of different cosolutes on in vitro enzyme kinetics yielded glimpses into their potential behavior when functioning in their natural, complex, in vivo milieu. Viewing cosolute in vitro influences on a model enzyme, calf intestinal alkaline phosphatase, as a combination of competitive and uncompetitive behaviors provided quantitative insights into their effects on catalysis. Observed decreases in the apparent specificity constant, K asp, caused by the presence of polyethylene glycols or betaine in the reaction solution, indicated interference with enzyme-substrate complex formation. This competitive inhibition appeared to be driven by osmotic stress. Dextran 6 K and sucrose strongly impeded the subsequent conversion of the bound substrate into a free product, which was marked by sharp reductions in V max, uncompetitive inhibition. For the same step, smaller noncarbohydrate cosolutes, triethylene glycol, polyethylene glycol 400, and betaine, also behaved as uncompetitive inhibitors but to a lesser extent. However, polyethylene glycol 8000 and 20,000 were uncompetitive activators, increasing V max. Polyethylene glycol of molecular weight 1000 displayed intermediate effects between these two groups of noncarbohydrate cosolutes. These results suggested that crowding has a strong influence on free product formation. The combination of competitive and uncompetitive effects and mixed behaviors, caused by the cosolutes on calf intestinal alkaline phosphatase kinetics, was consistent with the trends seen in similar enzyme-cosolute studies. It is proposed that the double-displacement mechanism of alkaline phosphatases, shared by many other enzymes, could be the root of this general observation.
| S-EPMC8515568 | biostudies-literature
Project description:Investigation of folding/unfolding DNA duplexes of various size and composition by superprecise calorimetry has revised several long-held beliefs concerning the forces responsible for the formation of the double helix. It was established that: 1) the enthalpy and the entropy of duplex unfolding are temperature dependent, increasing with temperature rise and having the same heat capacity increment for CG and AT pairs; 2) the enthalpy of AT melting is greater than that of the CG pair, so the stabilizing effect of the CG pair in comparison with AT results not from its larger enthalpic contribution (as expected from its extra hydrogen bond), but from the larger entropic contribution of the AT pair that results from its ability to fix ordered water in the minor groove and release it upon duplex unfolding; 3) the translation entropy, resulting from the appearance of a new kinetic unit on duplex dissociation, determines the dependence of duplex stability on its length and its concentration (it is an order-of-magnitude smaller than predicted from the statistical mechanics of gases and is fully expressed by the stoichiometric correction term); 4) changes in duplex stability on reshuffling the sequence (the "nearest-neighbor effect") result from the immobilized water molecules fixed by AT pairs in the minor groove; and 5) the evaluated thermodynamic components permit a quantitative expression of DNA duplex stability.
| S-EPMC5773757 | biostudies-literature
Project description:The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several enzymes and is often understood in terms of changes to enzyme conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3',5,5'-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with crowders and cosolutes. At high polyethylene glycol (PEG) concentrations (25-30 wt/wt %), no reaction was observed for TMB. Even at lower concentrations, Michaelis constants (KM) for HRP with the more hydrophobic substrate increased in the presence of crowding agents and cosolutes, particularly with PEG. Diffusion of TMB and OPD in the PEG and dextran reaction media was evaluated using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The diffusivity of the TMB decreased 3.9× in 10% PEG 8k compared to that in buffer and decreased only 1.7× for OPD. Together, these data suggest that weak attractive interactions between small-molecule substrates and crowders or cosolutes can reduce substrate chemical activity and consequently decrease enzyme activity and that these effects vary with the identity of the molecules involved. Because many enzymes can act on multiple substrates, it is important to consider substrate chemistry in understanding enzymatic reactions in complex media such as biological fluids.
| S-EPMC4161143 | biostudies-literature
Project description:The properties of non-canonical DNA structures, like G-quadruplexes and triplexes, change under cell-mimicking molecular crowding conditions relative to dilute aqueous solutions. The analysis of environmental effects on their stability is crucial since they play important roles in gene expression and regulation. In this study, three intramolecular and intermolecular triplex-forming sequences of different C+*G-C triplet content (*: Hoogsteen base pair; - : Watson-Crick base pair) were designed and their stability measured in the absence and presence of a crowding agent with different K+ concentrations. In dilute solution, the stability of the triplexes was reduced by decreasing the concentration of KCl. This reduction became smaller as the number of C+*G-C triplets increased. Under molecular crowding conditions, Watson-Crick base pairs and Hoogsteen base pairs were destabilized and stabilized, respectively. Interestingly, with lower KCl concentrations (≤1 M), the destabilization of the triplexes due to reduction of KCl concentration was significantly smaller than in dilute solutions. In addition, the C+*G-C content had greater influence on triplex stability under molecular crowding conditions. Our work provides quantitative information about the effects of K+ concentration on triplex stability under molecular crowding conditions and should further our understanding of the function and regulation of triplexes in bioprocesses.
| S-EPMC7024179 | biostudies-literature
Project description:The insertion of a DNA base moiety at the end of a DNA duplex to form a Watson-Crick or wobble pair during DNA annealing or replication is a step of fundamental biological importance. Therefore, we investigated the energetics of a formation of the terminal G x C, G x T, and G x A base pairs in DNA containing a 5'-dangling G adjacent to the base insertion point using differential scanning calorimetry and computer simulations. The energies calculated along classical molecular dynamics trajectories in aqueous solution were analyzed in the framework of linear-response approximation (LRA) to obtain relative free energies for the base insertion and their electrostatic, van der Waals, and preorganization components. Using the generic set of LRA parameters, the calculated free energies disfavored the mispair formation by 2.5 (G x C --> G x T) and 1.7 (G x C --> G x A) kcal/mol, in reasonable agreement with the experimental free energy differences of 1.8 and 1.4 kcal/mol, respectively. The calculated preorganization components of these free energies of 0.6 (G x C --> G x T) and -0.1 (G x C --> G x A) kcal/mol show that electrostatic preorganization, which is an important source of DNA replication fidelity, plays a lesser role in the mispair destabilization in the absence of DNA polymerase.
| S-EPMC2841231 | biostudies-literature