Origin of heat capacity increment in DNA folding: The hydration effect
Graphical abstract
Introduction
Studies of DNA energetics and its relation to DNA structures are one of the central topics in molecular biophysics. Early experiments have shown that upon heating double helix becomes disrupted and, in a highly cooperative transition, the two strands separate at elevated temperatures [1]. In contrast to proteins, this process is highly reversible. Energetics of DNA differs from that of proteins also in some other important aspects. For example, DNA stability is strongly affected by concentration of ions and may undergo a significant structural transitions in presence of certain cations [2]. Different alcohols strongly promote DNA duplex denaturation even at low concentrations [3]. Furthermore, as already noted in the early crystallographic experiments using dehydrated DNA samples adopting an unusual Z-DNA conformation, water activity strongly affects DNA conformation and its energetics [4]. One of the first links between the DNA sequence and its stability came from the observation that thermal stability depends linearly on the GC content [5]. This suggested that GC pairs are more stable than AT pairs and paved the road to prediction of duplex stability from the corresponding nucleotide sequence. In the following decades relationship between sequence and energetics has been delineated under the nearest-neighbor approach and increasingly refined parameters together with empirical relations enable sufficiently reliable prediction of DNA duplex melting temperatures [[6], [7], [8], [9]].
Complementary to these studies, significant advances have been made at explaining DNA energetics at the molecular level (reviewed in Privalov and Crane-Robinson, 2018 [10]). For example, it has been generally assumed that higher stability of GC pairs is due to the presence of three hydrogen bonds between bases whereas the AT is paired with only two hydrogen bonds. However, recent experiments have shown that stability is determined almost entirely by base-stacking interactions, while the correct hydrogen bonding of bases has a negligible effect on the stability and may even be destabilizing in case of AT pairs [11]. Calorimetric experiments have shown that formation of AT base pairs is enthalpically more favorable than GC pairs and this has been attributed to the presence of the bound water molecule in the AT minor groove [12]. Relationship between ion concentration and DNA stability can be successfully predicted using empirical relations and influence of ions is explained at the theoretical level by counter-ion condensation and Poisson-Boltzmann polyelectrolyte theories [8,13,14].
Heat capacity change ∆cp associated with biomolecular phase transitions is an extensively investigated quantity, particularly in the protein field. It influences the shape of protein stability curve and is crucial for prediction of its heat and cold denaturation. Initially, the existence of ∆cp, F associated with DNA folding has been overlooked, likely because the contribution is rather small in value. However, spectroscopic and calorimetric data from numerous studies as well as mechanical unfolding experiments have convincingly shown that DNA folding is accompanied by a small but significant negative change of ∆cp, F, thus having the same sign as for protein folding [[15], [16], [17], [18], [19]]. The specific heat capacity change associated with protein folding is on average − 0.53 J mol−1 g−1 while that for DNA folding it amounts only about −0.19 J mol−1 g−1. In a large survey of the literature Mikulecky and Feig found that majority of experimentally determined ∆cp, F values fall in range between −130 to −250 J mol−1 K−1 per base pair [20]. Rouzina and Bloomfield reanalyzed several published datasets and have arrived to approximately similar values, that is −170 to −400 J mol−1 K−1 [21]. Recent calorimetric experiments from Privalov group suggest that for short duplexes ∆cp, F contribution is −140 J mol−1 K−1 per base pair same for AT and GC pairs [12]. This value is practically identical to the ∆cp, F values determined in our laboratory earlier using global description of both calorimetric and spectroscopic data [22,23]. The exact magnitude of ∆cp, F contribution is important since it determines the temperature dependence of other thermodynamic parameters and it is therefore crucial for prediction of DNA stability over a wide temperature range.
Origin of the large heat capacity increment for protein folding has been extensively studied since it offers valuable information concerning the driving forces governing the folding process [24]. Even deeper insight can be gained when ∆cp, F is separated into contributions related to changes in electrostatics, solvation of the molecular surface and differences in the vibrational modes [25]. Because hydrophobic effect is considered one of the main driving forces for protein folding, many studies focused on the hydration heat capacity contribution ∆cphyd. Experiments using model compound data showed that burial of nonpolar atomic groups is accompanied by a negative change of the heat capacity while the reverse is true for the burial of polar groups [[26], [27], [28]]. These data as well as analysis of heat capacity increments for protein folding have been used to establish several empirical parametrizations relating changes in the solvent accessible surface area (ASA) and ∆cphyd [[27], [28], [29], [30], [31]]. It has been shown that the observed ∆cp, F for proteins is due mainly to the dehydration and by some estimates other contributions add only around 5–10% of the overall ∆cp, F change [32,33]. Thus, heat capacity changes related to solvent reorganization appear to explain ∆cp, F increment in proteins and the results from model compound studies significantly contributed to this view.
On the other hand, the origin of the heat capacity increment accompanying DNA folding is not understood. Surface-area models developed for proteins have been used to analyze heat capacity increments associated with DNA folding [15,34], ligand-DNA [[35], [36], [37], [38]] and protein-DNA binding [[39], [40], [41]] with mixed success. In many cases the observed ∆cp increment cannot be explained solely by the hydration contribution as calculated using surface area parametrizations developed for proteins. This suggests that either the hydration contribution is estimated incorrectly or that, in addition to hydration, some other components significantly contribute to the overall ∆cp. For example, it has been suggested that in protein folding coupled to DNA binding may explain large ∆cp increments [41]. Furthermore, it has been questioned whether surface area parametrizations developed for proteins can be applied directly to DNA systems [40]. To address this question Madan and Sharp estimated the hydration heat capacity of nucleic acid constituents from the solute induced changes of the water structure simulated by the Monte Carlo method [42]. Here we complement this theoretical study and analyze the experimental data of partial molar heat capacities of nucleic bases and nucleosides and calculate their hydration heat capacity contribution ∆cphyd. We then relate the obtained ∆cphyd to the ASA, and observe that empirical surface area coefficient for polar solvation clearly differs from that observed for the protein model compounds. Based on the burial of different DNA constituents upon folding we estimate the hydration heat capacity associated with DNA folding and discuss other potential contributions involved in this process. It appears that dehydration is one of the major sources to the observed overall ∆cp, F increment upon DNA folding, however other contributions are also significant and appear to be significantly compensated. A new parametrization is introduced to estimate ∆cp, Fhyd from the ∆ASA and the obtained values agree well with the experimental ∆cp, F increments measured for folding of B-DNA and noncanonical DNA structures.
Section snippets
Calculations of accessible surface area
All accessible surface area (ASA) calculations were preformed using NACCESS version 2.1.1. which uses a rolling probe method [43]. Default set of van der Waals atomic radii were used and the probe size was set to 1.4 Å. Hydrogen atoms were excluded from the calculation, C atoms were considered as nonpolar and N, O and P atoms as polar. ASA of nucleotides was calculated for the C3’ exo puckered form with base in the anti conformation. Average ASAU for nucleotides in the high-temperature unfolded
Partial molar heat capacities of nucleic acid constituents
First measurements of partial molar heat capacities of nucleic bases in aqueous solutions were reported by Kilday who studied solvation enthalpy as a function of temperature [51]. A more direct, calorimetric measurements were used in the subsequent studies, and were later expanded to the solutions of nucleosides [[52], [53], [54]]. Standard partial molar heat capacities for aqueous solutions of nucleic bases and nucleosides at 25 °C, as reported by Zielenkiewicz and Hedwig groups, are
Discussion
The origin and the magnitude of the hydration heat capacity contribution ∆cp, Fhyd associated with folding of DNA has not been studied in such great detail as one for protein folding. To fill this gap Madan and Sharp calculated ∆cphyd of nucleic bases from the configuration of water molecules in the first hydration shell as observed in the Monte Carlo simulation [42]. These ∆cphydvalues range from 75 to 130 J mol−1 K−1, which is in same range as reported here (40 to 135 J mol−1 K−1). Thus, the
Conclusion
Using the experimental data, we estimate the hydration heat capacity contributions ∆cphyd for DNA constituents. The obtained ∆cphyd values correlate with the total solvent accessible surface area. In contrast to results from protein model compounds we observe that polar ASA and ∆cphyd for nucleic bases and nucleosides are positively corelated. This suggests that polar hydration of nucleic bases differs from that of protein model compounds, explaining why surface parametrizations developed for
Author contributions
S. H. and J. L. designed the study and wrote the manuscript. S. H. analyzed the data.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was supported by the grants P1-0201 and J1-1706 from the Slovenian Research Agency. We are grateful to Uroš Zavrtanik for help with the preparation of the final version of the manuscript, to dr. Vojč Kocman for help with the MD simulations and to dr. Bojan Šarac for critical reading of the manuscript.
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