Denaturation Transition Research Articles

Computational prediction of the melting temperature of a DNA biosensor to detect Mycobacterium tuberculosis

Biosensors are DNA probes that form a stem-and-loop structure. When the biosensor binds to the complementary nucleic acid target, it undergoes a conformational transition that enables them to fluoresce brightly. Thermodynamic properties such as melting temperature ( T m) and free energy (Δ G) can to predict computationally the hybridization. The DNA denaturation temperature, also called the DNA melting temperature, is the temperature at which half of the DNA molecules are hybridized in double-helical structures, and half remain unhybridized. T m and Δ G yield information about the stability of the duplex. We modeled a biosensor with a sequence in the middle of the two primers that corresponds to the Mycobacterium tuberculosis complex in PCR solution conditions with AMPLITAQ. The hybridization thermodynamic properties were computed using a software package called the Oligonucleotide Modeling Platform (OMP; DNA Software, Inc.). The thermal denaturation and thermal transition profiles showed a melting temperature of 62 °C. We obtain a theoretical description of DNA denaturation by calculating the melting temperature of the complex. Our results are in good agreement with those obtained experimentally.

Dynamics of DNA melting

The dynamics of loops at the DNA denaturation transition is studied. A scaling argumentis used to evaluate the asymptotic behavior of the autocorrelation function of the state ofcomplementary bases (either open or closed). The long-time asymptotic behaviorof the autocorrelation function is expressed in terms of the entropy exponent,c, of a loop. The validity of the scaling argument is tested using a microscopic model of anisolated loop and a toy model of interacting loops. This suggests a method for measuringthe entropy exponent using single-molecule experiments such as fluorescence correlationspectroscopy.

Temperature and pressure dependence of protein stability: The engineered fluorescein-binding lipocalin FluA shows an elliptic phase diagram

We have measured the equilibrium constant for the denaturation transition of the engineered fluorescein-binding lipocalin FluA as a function of pressure and temperature, taking advantage of the fact that the ligand's fluorescence is almost fully quenched when complexed with the folded protein, but reversibly reappears on denaturation. From the equilibrium constant as a function of pressure and temperature all of the involved thermodynamic parameters of protein folding, in particular the changes in entropy and volume, compressibility, thermal expansion, and specific heat, were deduced in a global fitting procedure. Assuming that these parameters are independent of temperature and pressure, we can demonstrate from the ratio of Deltabeta, Deltaalpha(2), DeltaC(p) that the phase diagram of protein folding assumes an elliptic shape. Furthermore, we can show that the thermodynamic condition for such an elliptic phase diagram is related to the degree of correlation between the fluctuations of the changes in volume and enthalpy at the phase boundary. For the protein investigated this correlation is low, as generally expected for highly degenerate systems. Our study suggests that the elliptic phase diagram is a consequence of the inherent conformational disorder of proteins and that it may be viewed as the thermodynamic manifestation of the high degeneracy of conformational energies that is characteristic for this class of macromolecules.

Simplified Langevin approach to the Peyrard-Bishop-Dauxois model of DNA

A simple Langevin approach is used to study stationary properties of the Peyrard-Bishop-Dauxois model for DNA, allowing known properties to be recovered in an easy way. Results are shown for the denaturation transition in homogeneous samples, for which some implications, so far overlooked, of an analogy with equilibrium wetting transitions are highlighted. This analogy implies that the order parameter, asymptotically, exhibits a second-order transition even if it may be very abrupt for nonzero values of the stiffness parameter. Not surprisingly, we also find that, for heterogeneous DNA, within this model the largest bubbles in the premelting stage appear in adenine-thymine-rich regions, while we suggest the possibility of some sort of not strictly local effects owing to the merging of bubbles.

Z2 Symmetry in the Melting Transition of the DNA Helix

The denaturation transition of DNA molecules is numerically investigated with a focus on the Z2 symmetry related with the two opposite directions of the winding: the left-handed (Z-type) and the right-handed (B-type) windings. We extend the previous DNA model Hamiltonian to include a term that phenomenologically describes the tangential interaction, making the same twist direction favored by adjacent base pairs. Evoking the large strength of the tangential interaction, we reveal the Z2 symmetry nature of twist directions associated with the opening of DNA strands. We also observe that the boundary between the B-domain and the Z-domain is point-like, in accordance with a recent experimental result.

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min − 1 the denaturation temperature of PA at pH 6.0 decreases by about 6 °C, while the denaturation enthalpy does not change notably giving an average value of 31.6 ± 2.1 J g − 1 . The denaturation temperature of PA reaches a maximum value of 64.5 °C at pH 6.0 and decreases by about of 15 °C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58 ± 0.02 J g − 1 K − 1 . The denaturation transition of PA is approximated by the Lumry–Eyring model. The first stage of the process is assumed to be a reversible unfolding of the α-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the β-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the α-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the α-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.

Apo‐parvalbumin as an intrinsically disordered protein

Recently defined family of intrinsically disordered proteins (IDP) includes proteins lacking rigid tertiary structure meanwhile fulfilling essential biological functions. Here we show that apo-state of pike parvalbumin (alpha- and beta-isoforms, pI 5.0 and 4.2, respectively) belongs to the family of IDP, which is in accord with theoretical predictions. Parvalbumin (PA) is a 12-kDa calcium-binding protein involved into regulation of relaxation of fast muscles. Differential scanning calorimetry measurements of metal-depleted form of PA revealed the absence of any thermally induced transitions with measurable denaturation enthalpy along with elevated specific heat capacity, implying the lack of rigid tertiary structure and exposure of hydrophobic protein groups to the solvent. Calcium removal from the PAs causes more than 10-fold increase in fluorescence intensity of hydrophobic probe bis-ANS and is accompanied by a decrease in alpha-helical content and a marked increase in mobility of aromatic residues environment, as judged by circular dichroism spectroscopy (CD). Guanidinium chloride-induced unfolding of the apo-parvalbumins monitored by CD showed the lack of fixed tertiary structure. Theoretical estimation of energetics of the charge-charge interactions in the PAs indicated their pronounced destabilization upon calcium removal, which is in line with sequence-based predictions of disordered protein chain regions. Far-UV CD studies of apo-alpha-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20 degrees C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10-15 degrees C is revealed by near-UV CD for both PAs. The absence of detectable enthalpy change in this temperature region suggests continuous nature of the transition. Overall, the theoretical and experimental data obtained show that PA in apo-state is essentially disordered nevertheless demonstrates complex denaturation behavior. The native rigid tertiary structure of PA is attained upon association of one (alpha-PA) or two (beta-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data.

A breathing wormlike chain model on DNA denaturation and bubble: Effects of stacking interactions

DNA stably exists as a double-stranded structure due to hydrogen-bonding and stacking interactions between bases. The stacking interactions are strengthened when DNA is paired, which results in great enhancement of bending rigidity. We study the effects of this stacking-induced stiffness difference on DNA denaturation and bubble formations. To this end, we model double-stranded DNA as a duplex of two semiflexible chains whose persistence length varies depending on the base-pair distance. Using this model, we perform the Langevin dynamics simulation to examine the characteristics of the denaturation transition and the statistics of the bubbles. We find that the inclusion of the stacking interactions causes the denaturation transition to be much sharper than otherwise. At physiological temperature, the stacking interactions prohibit the initiation of bubble formation but promote bubbles, once grown, to retain the large size.

Thermal denaturation of fluctuating finite DNA chains: The role of bending rigidity in bubble nucleation

Statistical DNA models available in the literature are often effective models where the base-pair state only (unbroken or broken) is considered. Because of a decrease by a factor of 30 of the effective bending rigidity of a sequence of broken bonds, or bubble, compared to the double stranded state, the inclusion of the molecular conformational degrees of freedom in a more general mesoscopic model is needed. In this paper we do so by presenting a one-dimensional Ising model, which describes the internal base-pair states, coupled to a discrete wormlike chain model describing the chain configurations [J. Palmeri, M. Manghi, and N. Destainville, Phys. Rev. Lett. 99, 088103 (2007)]. This coupled model is exactly solved using a transfer matrix technique that presents an analogy with the path integral treatment of a quantum two-state diatomic molecule. When the chain fluctuations are integrated out, the denaturation transition temperature and width emerge naturally as an explicit function of the model parameters of a well defined Hamiltonian, revealing that the transition is driven by the difference in bending (entropy dominated) free energy between bubble and double-stranded segments. The calculated melting curve (fraction of open base pairs) is in good agreement with the experimental melting profile of poly(dA)-poly(dT) and, by inserting the experimentally known bending rigidities, leads to physically reasonable values for the bare Ising model parameters. Among the thermodynamical quantities explicitly calculated within this model are the internal, structural, and mechanical features of the DNA molecule, such as bubble correlation length and two distinct chain persistence lengths. The predicted variation of the mean-square radius as a function of temperature leads to a coherent explanation for the experimentally observed thermal viscosity transition. Finally, the influence of the DNA strand length is studied in detail, underlining the importance of finite size effects, even for DNA made of several thousand base pairs. Simple limiting formulas, useful for analyzing experiments, are given for the fraction of broken base pairs, Ising and chain correlation functions, effective persistence lengths, and chain mean-square radius, all as a function of temperature and DNA length.

Denaturation Transition of Stretched DNA

We generalize the Poland-Scheraga model to consider DNA denaturation in the presence of an external stretching force. We demonstrate the existence of a force-induced DNA denaturation transition and obtain the temperature-force phase diagram. The transition is determined by the loop exponent c, for which we find the new value c = 4 nu-1/2 such that the transition is second order with c = 1.85 < 2 in d = 3. We show that a finite stretching force F destabilizes DNA, corresponding to a lower melting temperature T(F), in agreement with single-molecule DNA stretching experiments.

Relationship between the Stability of Hen Egg-White Lysozymes Mutated at Sites Designed to Interact with α-Helix Dipoles and Their Secretion Amounts in Yeast

The positively charged lysine at the C-terminals of three long alpha-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change Delta G of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long alpha-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and Delta G of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long alpha-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.

Kinetically controlled refolding of a heat-denatured hyperthermostable protein

The thermal denaturation of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144 degrees C, corresponding to protein denaturation and complete unfolding, respectively, as shown by circular dichroism and fluorescence spectroscopy data. Calorimetric studies also showed that the denatured state did not refold to the native state unless the cooling temperature rate was very slow. Furthermore, previously denatured protein samples gave well-resolved denaturation transition peaks and showed enzymatic activity after 3 and 9 months of storage, indicating slow refolding to the native conformation over time.

Thermal Denaturation of Fluctuating DNA Driven by Bending Entropy

A statistical model of homopolymer DNA, coupling internal base-pair states (unbroken or broken) and external thermal chain fluctuations, is exactly solved using transfer kernel techniques. The dependence on temperature and DNA length of the fraction of denaturation bubbles and their correlation length is deduced. The thermal denaturation transition emerges naturally when the chain fluctuations are integrated out and is driven by the difference in bending (entropy dominated) free energy between broken and unbroken segments. Conformational properties of DNA, such as persistence length and mean-square-radius, are also explicitly calculated, leading, e.g., to a coherent explanation for the experimentally observed thermal viscosity transition.

Membrane proteins in thin films

Molecular functions and structural changes of membrane proteins in an aqueous environment can be elucidated by reaction-induced FTIR difference spectroscopy upon photolysis of caged compounds. The achieved detection of IR band changes even due to single amino acid residues is, however, only possible in the presence of very high protein concentrations, implying that a low water content must be present. In general, the films are formed by controlled dehydration of membrane protein suspensions at reduced pressure and low temperature. For the retention of enzymatic activity of Na,K-ATPase, for example, a cosolvent such as glycerol is required. In order to interprete the results obtained by FTIR spectroscopy, it is important to know whether essential properties of the proteins such as hydration are changed upon film formation. Therefore, a differential scanning calorimetry (DSC) study has been carried out with purified Na,K-ATPase and Ca-ATPase in suspension, in form of pellets obtained by high-speed ultracentrifugation and in thin films. As relevant thermoanalytical properties, the endothermic denaturation transitions of the proteins have been studied.

Beyond Lumry–Eyring: An unexpected pattern of operational reversibility/irreversibility in protein denaturation

We have found that, contrary to naïve intuition, the degree of operational reversibility in the thermal denaturation of lipase from Thermomyces lanuginosa (an important industrial enzyme) in urea solutions is maximum when the protein is heated several degrees above the end of the temperature-induced denaturation transition. Upon cooling to room temperature, the protein seems to reach a state with enzymatic activity similar to that of the initial native state, but with higher denaturation temperature and radically different behavior in terms of susceptibility to irreversible denaturation. These results show that patterns of operational reversibility/irreversibility in protein denaturation may be more complex than the often-taken-for-granted, two-situation classification (reversible vs. irreversible). Furthermore, they are consistent with the possibility of existence of different native or native-like states separated by high kinetic barriers under native conditions and they suggest experimental procedures to reach and study such "alternative" native states.

Reaction Rate Calculation by Parallel Path Swapping

The efficiency of path sampling simulations can be improved considerably using the approach of path swapping. For this purpose, we devise a new algorithmic procedure based on the transition interface sampling technique. In the same spirit of parallel tempering, paths between different ensembles are swapped, but the role of temperature is here played by the interface position. We test the method on the denaturation transition of DNA using the Peyrard-Bishop-Dauxois model. We find that the new algorithm gives a reduction of the computational cost by a factor of 20.

DNA binding properties and evaluation of cytotoxic activity of 9,10-bis- N-substituted (aminomethyl)anthracenes

The results of DNA binding properties for four selected N-substituted 9,10-bis(aminomethyl)anthracenes are presented. DNA binding affinities were studied using UV–vis and fluorescence spectrophotometric titrations, CD spectroscopy, denaturation transition temperature ( T m) measurements and AM1 quantum chemical calculations. The results obtained indicate that the anthracene products intercalate into the stacked base pairs of DNA with binding constants, K, in the range 1.3–10.9 × 10 5 M −1 and the binding site size in DNA-base pairs, n, extending over the range 2.4–4.6. T m values increased in the presence of the anthryl probes, thereby reflecting an increased stability of the calf-thymus (CT) DNA double helix and rendering agreement with the spectrometric titration results. The synthesized compounds were tested against L1210 and HeLa tumor cell lines wherein the HeLa cells appeared to be more sensitive than the L1210 cells. 9,10-Bis{[2-(piperazin-1-yl)ethyl]aminomethyl}anthracene exhibited the highest activity of the tested compounds. Our findings were compared with those of a control drug bisantrene.

The helix–turn–helix motif as an ultrafast independently folding domain: The pathway of folding of Engrailed homeodomain

Helices 2 and 3 of Engrailed homeodomain (EnHD) form a helix-turn-helix (HTH) motif. This common motif is believed not to fold independently, which is the characteristic feature of a motif rather than a domain. But we found that the EnHD HTH motif is monomeric and folded in solution, having essentially the same structure as in full-length protein. It had a sigmoidal thermal denaturation transition. Both native backbone and local tertiary interactions were formed concurrently at 4 x 10(5) s(-1) at 25 degrees C, monitored by IR and fluorescence T-jump kinetics, respectively, the same rate constant as for the fast phase in the folding of EnHD. The HTH motif, thus, is an ultrafast-folding, natural protein domain. Its independent stability and appropriate folding kinetics account for the stepwise folding of EnHD, satisfy fully the criteria for an on-pathway intermediate, and explain the changes in mechanism of folding across the homeodomain family. Experiments on mutated and engineered fragments of the parent protein with different probes allowed the assignment of the observed kinetic phases to specific events to show that EnHD is not an example of one-state downhill folding.

Loop Dynamics in DNA Denaturation

The dynamics of a loop in DNA molecules at the denaturation transition is studied by scaling arguments and numerical simulations. The autocorrelation function of the state of complementary bases (either closed or open) is calculated. The long-time decay of the autocorrelation function is expressed in terms of the loop exponent c both for homopolymers and heteropolymers. This suggests an experimental method for measuring the exponent c using florescence correlation spectroscopy.

DSC study of the association of ethanol with human serum albumin

Human serum albumin unfolding in ethanol/water mixtures was studied by use of differential scanning calorimetry. Ethanol-induced changes in DSC curves of defatted and non-defatted albumin were markedly different. In the presence of ethanol, bimodal denaturation transition for fatty acid free albumin was observed while that for albumin containing endogenous fatty acids was single and more sharpen than in aqueous solution. Ethanol was found to decrease the thermal stability of albumin due to the binding to the unfolded state to a higher degree than to the native state, thus favouring unfolding. The binding with different affinities has been suggested depending on ethanol concentration range.