On the truncation of long-range electrostatic interactions in DNA

Long-range interactions are known to play an important role in highly polar biomolecules like DNA. In molecular dynamics simulations of nucleic acids and proteins, an accurate treatment of the long-range interactions are crucial for achieving stable nanosecond trajectories. In this report, we evaluate the structural and dynamic effects on a highly charged oligonucleotide in aqueous solution from different long-range truncation methods. Two group-based truncation methods, one with a switching function and one with a force-switching function were found to fail to give accurate stable trajectories close to the crystal structure. For these group-based truncation methods, large root mean square (rms) deviations from the initial structure were obtained and severe distortions of the oligonucleotide were observed. Another group-based truncation scheme, which used an abrupt truncation at 8. 0 A or at 12.0 A was also investigated. For the short cutoff distance, the conformations deviated far away from the initial structure and were significantly distorted. However, for the longer cutoff, where all necessary electrostatic interactions were included, the trajectory was quite stable. For the particle mesh Ewald (PME) truncation method, a stable DNA simulation with a heavy atom rms deviation of 1.5 A was obtained. The atom-based truncation methods also resulted in stable trajectories, according to the rms deviation from the initial B-DNA structure, of between 1.5 and 1.7 A for the heavy atoms. In these stable simulations, the heavy atom rms deviations were approximately 0.6-1.0 A lower for the bases than for the backbone. An increase of the cutoff radius from 8 to 12 A decreased the rms deviation by approximately 0.2 A for the atom-based truncation method with a force-shifting function, but increased the computational time by a factor of 2. Increasing the cutoff from 12 to 18 A for the atom-based truncation method with a force-shifting function requires 2-3 times more computational time, but did not significantly change the rms deviation. Similar rms deviations from the initial structure were found for the atom-based method with a force-shifting function and for the PME method. The computational cost was longer for the PME method with a cutoff of 12. 0 A for the direct space nonbonded calculations than for the atom-based truncation method with a force-shifting function and a cutoff of 12.0 A. If a nonperiodic boundary, e.g., a spherical boundary, was used, a considerable speedup could be achieved. From the rms fluctuations, the terminal nucleotides and especially the cytidines were found to be more flexible than the nonterminal nucleotides. The B-DNA form of the oligonucleotide was maintained throughout the simulations and is judged to depend on the parameters of the energy function and not on the truncation method used to handle the long-range electrostatic interactions. To perform accurate and stable simulations of highly charged biological macromolecules, we recommend that the atom-based force-shift method or the PME method should be used for the long-range electrostatics interactions.     

Comparison of different schemes to treat long-range electrostatic interactions in molecular dynamics simulations of a protein crystal

Eight molecular dynamics simulations of a ubiquitin crystal unit cell were performed to investigate the effect of different schemes to treat the long-range electrostatic interactions as well as the need to include counter ions. A crystal system was chosen as the test system, because the higher charge density compared with a protein in solution makes it more sensitive to the way of treating the electrostatic interactions. Three different schemes of treating the long-range interactions were compared: straight cutoff, reaction-field approximation, and a lattice-sum method (P3M). For each of these schemes, two simulations were performed, one with and one without the counter ions. Two additional simulations with a reaction-field force and different initial placements of the counter ions were performed to examine the effect of the initial positions of the ions. The inclusion of long-range electrostatic interactions using either a reaction-field or a lattice-sum method proved to be necessary for the simulation of crystals. These two schemes did not differ much in their ability to reproduce the crystallographic structure. The inclusion of counter ions, on the other hand, seems not necessary for obtaining a stable simulation. The initial positions of the ions have a visible but small effect on the simulation.     

Thermal stability and DNA binding activity of a variant form of the Sso7d protein from the archeon Sulfolobus solfataricus truncated at leucine 54

Sso7d is a 62-residue, basic protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Around neutral pH, it exhibits a denaturation temperature close to 100 degrees C and a non-sequence-specific DNA binding activity. Here, we report the characterization by circular dichroism and fluorescence measurements of a variant form of Sso7d truncated at leucine 54 (L54Delta). It is shown that L54Delta has a folded conformation at neutral pH and that its thermal unfolding is a reversible process, represented well by the two-state N <=> D transition model, with a denaturation temperature of 53 degrees C. Fluorescence titration experiments indicate that L54Delta binds tightly to calf thymus DNA, even though the binding parameters are smaller than those of the wild-type protein. Therefore, the truncation of eight residues at the C-terminus of Sso7d markedly affects the thermal stability of the protein, which nevertheless retains a folded structure and DNA binding activity.     

Molecular dynamics simulation and experimental validation of the effect of pH on protein desorption in hydrophobic charge induction chromatography

The ligands in hydrophobic charge induction chromatography (HCIC) are hydrophobic and ionisable. Thus, the pH is crucial for the separation performance in HCIC, especially for elution. However, it is difficult to obtain the microscopic information in HCIC through experimental means. In this work, molecular dynamics simulations are performed to examine the effect of pH on elution and protein conformational transition in HCIC, using a 46-bead β-barrel coarse-grained model protein and an HCIC adsorbent pore model constructed in an earlier work. Corresponding experiments are carried out for the validation of simulation results, using lysozyme and MEP Hypercel. Both the activities and fluorescence of lysozyme are examined to evaluate the conformational transition. The simulations indicate that the elution efficiency of protein increases with decreasing pH value in a non-linear manner. This is qualitatively consistent with the experimental results. MD simulations indicate that protein unfolding occurs in elution at all pH values. However, the experimental data show that the activity and conformation of lysozyme is independent of pH of the elution buffer. The microscopic information from simulation shows that protein unfolding is mainly observed on the adsorbent surface, but it cannot be detected in the experiments that only probe the proteins in the bulk liquid.     

Tracking the unfolding and refolding pathways of outer membrane protein porin from Paracoccus denitrificans

We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin.     

Unfolding of the cold shock protein studied with biased molecular dynamics

The cold shock protein from Bacillus caldolyticus is a small beta-barrel protein that folds in a two-state mechanism. For the native protein and for several mutants, a wealth of experimental data are available on stability and folding, so that it is an optimal system to study this process. We compare data from unfolding simulations (trajectories of 5 and up to 12 ns) obtained with a bias potential at room temperature and from unbiased thermal unfolding simulations with experimental data. The unfolding patterns derived from the trajectories starting from different native-like conformations and subject to different unfolding conditions agree. The transition state found in the simulations of unfolding is close to the native structure in agreement with experiment. Moreover, a lower value of the free energy barrier of unfolding was found for the mutant R3E than for the mutant E46A and the native protein, as indicated by experimental data. The first unfolding event involves the three-stranded beta-sheet whose decomposition corresponds to the transition state. In contrast to conclusions drawn from experiments, we found that the two-stranded beta-strand forms the most stable substructure, which decomposes very late in the unfolding process. However, assuming that this structure forms very early in the folding process, our findings would not contradict the experiments but require a different interpretation of them.     

Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant.

We have studied the stability of the histone-like, DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant by differential scanning microcalorimetry and CD under acidic conditions at various concentrations within the range of 2-225 micro m of monomer. The thermal unfolding of both proteins is highly reversible and clearly follows a two-state dissociation/unfolding model from the folded, dimeric state to the unfolded, monomeric one. The unfolding enthalpy is very low even when taking into account that the two disordered DNA-binding arms probably do not contribute to the cooperative unfolding, whereas the quite small value for the unfolding heat capacity change (3.7 kJ.K(-1).mol(-1)) stabilizes the protein within a broad temperature range, as shown by the stability curves (Gibbs energy functions vs. temperature), even though the Gibbs energy of unfolding is not very high either. The protein is stable at pH 4.00 and 3.75, but becomes considerably less so at pH 3.50 and below, to the point that a simple decrease in concentration will lead to unfolding of both the wild-type and the mutant protein at pH 3.50 and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more stable than its E34D mutant, particularly at pH 4.00 and 3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJ.mol(-1) at 25 degrees C for DeltaDeltaG at pH 4.00, 3.75 and 3.50, respectively), which seems to be related to the effect of a salt bridge between E34 and K13.     

Effect of sol–gel encapsulation on the unfolding of ferric horse heart cytochrome c

Electronic absorption and resonance Raman spectra of ferric cytochrome c embedded in wet silica gels, in the presence of guanidine HCl as unfolding agent, between pH 0.35 and 7.0 are presented. The data clearly show that the ferric form of the protein encapsulated in sol–gel preserves its active site conformation. However, the spectra of the unfolded embedded protein are different from the corresponding spectra in solution suggesting that a strong interaction between the protein and the sol–gel takes place upon unfolding. The unfolding process mainly depends on the interaction between the exposed positive charges of the unfolded protein and the negatively charged functional groups of the silica surfaces. While this interaction partially stabilizes the protein in its native structure even at very acidic pH, in the presence of denaturants it has the opposite effect, causing mainly the weakening of both the heme-protein and the heme-ligand interactions.     

Molecular dynamics simulation reveals a surface salt bridge forming a kinetic trap in unfolding of truncated Staphylococcal nuclease

Surface salt bridges are ubiquitous in globular proteins. Their contribution to protein stability has been extensively debated in the past decade. Here, molecular dynamics simulations are performed starting from a non-equilibrium state of Staphylococcal nuclease (SNase) with C-terminal truncation (SNaseDelta). The results indicate a key role in the unfolding of the surface salt bridge between arginine 105 and glutamate 135. Experimentally, SNaseDelta is known to be partially unfolded. However, in simulations over 1 ns at 300 K and over 500 ps at 400 K, SNaseDelta remains stable in the native-like folded conformation, the salt bridge hindering unfolding. When the potential function is altered so as to selectively weaken the salt bridge, which then breaks rapidly at 430 K, the protein starts to unfold. The results suggest that breaking of this salt bridge presents a significant barrier to the unfolding transition of SNaseDelta from a native-like state to the unfolded state. Potential of mean force calculations indicate that the barrier height for this transition is approximately 7 kcal/mol.     

Urea and guanidinium chloride denature protein L in different ways in molecular dynamics simulations

In performing protein-denaturation experiments, it is common to employ different kinds of denaturants interchangeably. We make use of molecular dynamics simulations of Protein L in water, in urea, and in guanidinium chloride (GdmCl) to ascertain if there are any structural differences in the associated unfolding processes. The simulation of proteins in solutions of GdmCl is complicated by the large number of charges involved, making it difficult to set up a realistic force field. Furthermore, at high concentrations of this denaturant, the motion of the solvent slows considerably. The simulations show that the unfolding mechanism depends on the denaturing agent: in urea the β-sheet is destabilized first, whereas in GdmCl, it is the α-helix. Moreover, whereas urea interacts with the protein accumulating in the first solvation shell, GdmCl displays a longer-range electrostatic effect that does not perturb the structure of the solvent close to the protein.     

Chitosan-soyprotein interaction as determined by thermal unfolding experiments

Chitosan interaction with soybean beta-conglycinin beta(3) was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (T(m)) was 63.4 degrees C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the T(m) value was elevated by 7.7 degrees C, whereas the T(m) elevation upon addition of chitosan hexamer (GlcN)(6) was 2.2 degrees C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the T(m) elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the T(m) elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with beta-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.     

Charge requirements for proton gradient-driven translocation of anthrax toxin

Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (ΔpH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ΔpH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ΔpH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LF(N)) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LF(N) to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LF(N) translocate independently of the ΔpH driving force. Acidic residues markedly increase the rate of ΔpH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ΔpH-driven unfolding and translocation. We discuss a ΔpH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the "molecular teeth" of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ΔpH translocation.     

Molecular dynamics simulations of human prion protein: importance of correct treatment of electrostatic interactions.

Molecular dynamics simulations have been used to investigate the dynamical and structural behavior of a homology model of human prion protein HuPrP(90-230) generated from the NMR structure of the Syrian hamster prion protein ShPrP(90-231) and of ShPrP(<90-231) itself. These PrPs have a large number of charged residues on the protein surface. At the simulation pH 7, HuPrP(90-230) has a net charge of -1 eu from 15 positively and 14 negatively charged residues. Simulations for both PrPs, using the AMBER94 force field in a periodic box model with explicit water molecules, showed high sensitivity to the correct treatment of the electrostatic interactions. Highly unstable behavior of the structured region of the PrPs (127-230) was found using the truncation method, and stable trajectories could be achieved only by including all the long-range electrostatic interactions using the particle mesh Ewald (PME) method. The instability using the truncation method could not be reduced by adding sodium and chloride ions nor by replacing some of the sodium ions with calcium ions. The PME simulations showed, in accordance with NMR experiments with ShPrP and mouse PrP, a flexibly disordered N-terminal part, PrP(90-126), and a structured C-terminal part, PrP(127-230), which includes three alpha-helices and a short antiparallel beta-strand. The simulations showed some tendency for the highly conserved hydrophobic segment PrP(112-131) to adopt an alpha-helical conformation and for helix C to split at residues 212-213, a known disease-associated mutation site (Q212P). Three highly occupied salt bridges could be identified (E146/D144<-->R208, R164<-->D178, and R156<-->E196) which appear to be important for the stability of PrP by linking the stable main structured core (helices B and C) with the more flexible structured part (helix A and strands A and B). Two of these salt bridges involve disease-associated mutations (R208H and D178N). Decreased PrP stability shown by protein unfolding experiments on mutants of these residues and guanidinium chloride or temperature-induced unfolding studies indicating reduced stability at low pH are consistent with stabilization by salt bridges. The fact that electrostatic interactions, in general, and salt bridges, in particular, appear to play an important role in PrP stability has implications for PrP structure and stability at different pHs it may encounter physiologically during normal or abnormal recycling from the pH neutral membrane surface into endosomes or lysomes (acidic pHs) or in NMR experiments (5.2 for ShPrP and 4.5 for mouse PrP).     

The mechanism of the amyloidogenic conversion of T7 endonuclease I

Amyloid fibrils are associated with a range of human disorders. Understanding the conversion of amyloidogenic proteins from their soluble forms to amyloid fibrils is critical for developing effective therapeutics. Previously we showed that T7 endonuclease I forms amyloid-like fibrils. Here we study the mechanism of the amyloidogenic conversion of T7 endonuclease I. We show that T7 endonuclease I forms fibrils at pH 6.8, but not at pH 6.0 or 8.0. The amyloidogenicity at pH 6.8 is not correlated with thermodynamic stability, unfolding cooperativity, or solubility. Thermal melting experiments at various pH values show that the protein has a distinctive thermal transition at pH 6.8. The transition at pH 6.8 has a lower transition temperature than the unfolding transitions observed at pH 6.0 and 8.0 and leads to a beta-rich conformation instead of an unfolded state. Electron microscopy shows that the thermal transition at pH 6.8 results in fibril formation. The thermal transition at pH 6.8 leads to a protein state that is not accessible at pH 6.0 or 8.0, showing that the existence of the amyloidogenic conformation of T7 endonuclease I depends sensitively on solution conditions. Therefore, we propose that fibrillizing proteins need to be "prepared" for fibrillization. Preparation may consist of amino acid replacements or changing solution conditions and may require retention of some aspects of native structure. In this model, some amyloid-enhancing mutations decrease protein stability, whereas others have little effect.     

Equilibrium dissociation and unfolding of the Arc repressor dimer

The equilibrium unfolding reaction of Arc repressor, a dimeric DNA binding protein encoded by bacteriophage P22, can be monitored by fluorescence or circular dichroism changes. The stability of Arc is concentration dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The stability of the protein is decreased at low pH and increased by high salt concentration. The salt dependence suggests that two ions bind preferentially to the folded protein. In 10 mM potassium phosphate (pH 7.3) and 100 mM KCl, the unfolding free energy reaches a maximum near room temperature. The results suggest that at the low protein concentrations where operator DNA binding is normally measured, Arc is predominantly monomeric and unfolded.     

Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.     

Explicit-solvent molecular dynamics simulations of a DNA tetradecanucleotide duplex: lattice-sum versus reaction-field electrostatics

Five long-timescale (10 ns) explicit-solvent molecular dynamics simulations of a DNA tetradecanucleotide dimer are performed using the GROMOS 45A4 force field and the simple-point-charge water model, in order to investigate the effect of the treatment of long-range electrostatic interactions as well as of the box shape and size on the structure and dynamics of the molecule (starting from an idealised B-DNA conformation). Long-range electrostatic interactions are handled using either a lattice-sum (LS) method (particle–particle–particle–mesh; one simulation performed within a cubic box) or a cutoff-based reaction-field (RF) method (four simulations, with long-range cutoff distances of 1.4 or 2.0 nm and performed within cubic or truncated octahedral periodic boxes). The overall double-helical structure, including Watson–Crick (WC) base-pairing, is well conserved in the simulation employing the LS scheme. In contrast, the WC base-pairing is nearly completely disrupted in the four simulations employing the RF scheme. These four simulations result in highly distorted compact (cutoff distance of 1.4 nm) or extended (cutoff distance of 2 nm) structures, irrespective of the shape and size of the computational box. These differences observed between the two schemes seem correlated with large differences in the radial distribution function between charged entities (backbone phosphate groups and sodium counterions) within the system.     

Pressure denaturation of staphylococcal nuclease studied by neutron small-angle scattering and molecular simulation

We studied the pressure-induced folding/unfolding transition of staphylococcal nuclease (SN) over a pressure range of approximately 1-3 kilobars at 25 degrees C by small-angle neutron scattering and molecular dynamics simulations. We find that applying pressure leads to a twofold increase in the radius of gyration derived from the small-angle neutron scattering spectra, and P(r), the pair distance distribution function, broadens and shows a transition from a unimodal to a bimodal distribution as the protein unfolds. The results indicate that the globular structure of SN is retained across the folding/unfolding transition although this structure is less compact and elongated relative to the native structure. Pressure-induced unfolding is initiated in the molecular dynamics simulations by inserting water molecules into the protein interior and applying pressure. The P(r) calculated from these simulations likewise broadens and shows a similar unimodal-to-bimodal transition with increasing pressure. The simulations also reveal that the bimodal P(r) for the pressure-unfolded state arises as the protein expands and forms two subdomains that effectively diffuse apart during initial stages of unfolding. Hydrophobic contact maps derived from the simulations show that water insertions into the protein interior and the application of pressure together destabilize hydrophobic contacts between these two subdomains. The findings support a mechanism for the pressure-induced unfolding of SN in which water penetration into the hydrophobic core plays a central role.     

Worm-like Ising model for protein mechanical unfolding under the effect of osmolytes

We show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte.