Single-molecule study on conformational dynamics of M.HhaI

We found that apo DNA methyltransferase M.HhaI under the physiological salt concentration does not possess the structure characterized by X-ray crystallography; instead, it interchanges between prefolded and unfolded states. Only after binding to the substrate, it transforms into a crystal-structure-like state. Flipping rates of its catalytic loop were directly measured.

Huge conformational rearrangements in M.HhaI were observed by a single-molecule study.

DNA methylation is an essential epigenetic mark, which plays regulatory roles in cell development and disease.¹ A thorough understanding of the DNA methylation mechanism is important to exploit epigenetics and also to guide drug design. Bacterial DNA methyltransferase M.HhaI is the first DNA methyltransferase whose structure was determined using X-ray crystallography.² It recognizes and methylates the inner cytosine of GCGC target sites in duplex DNA in the presence of cofactor S-adenosylmethionine (AdoMet), which transforms into S-adenosylhomocysteine (AdoHcy) after the reaction. Structural studies can provide valuable clues to understand how the enzyme executes its catalytic function. By comparing the crystal structures of M.HhaI with and without DNA, an essential catalytic loop (residues 80–100) in M.HhaI is observed to flip from the open to closed state along with a 180° turn of the target base. These motions adjust the target base into a proper position for methylation and have been under intensive studies.^3–6 The flipping motion of the catalytic loop is an essential part of the catalytic process, but its kinetic information was only inferred indirectly and its actual rates remain to be determined.^4–6Apart from the catalytic loop reorganization, M.HhaI may experience global rearrangement upon substrate binding. Molecular dynamics (MD) simulations showed the anti-correlated motions of residues separated by 3 nm, indicating domain compression during catalysis.⁷ NMR studies suggested that M.HhaI is a dynamic protein⁸ and that there are dramatic chemical shift perturbations reaching many distal sites upon DNA binding.⁹ Due to the lack of direct access to tertiary structures, they could not differentiate whether these distal changes result from structural adjustments from the crystal structure at the local level or concerted, global structural changes.To answer all the above questions, in this study, we used single-molecule techniques, i.e., single-molecule fluorescence resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS) to study the conformational dynamics of M.HhaI. smFRET, well known as ‘spectroscopic ruler’, which has been successfully used to study many fundamental biological mechanisms¹⁰ and has long-term progress and applications in structural biology.¹¹ FCS can detect the dynamics of transition under equilibrium condition.¹² With time resolution as high as nanoseconds, it can detect a process that is too fast to be studied by ensemble methods. By applying smFRET and FCS techniques, we directly measured the flipping rates of the catalytic loop and revealed the global conformational reorganizations of M.HhaI. Surprisingly, we found that M.HhaI alone (apo M.HhaI) under the physiological salt concentration did not possess the structure characterized by X-ray crystallography. Instead, it interchanges between prefolded and unfolded states. Only after it binds to the substrate, M.HhaI turns into its crystal-structure-like state.First, we probed the global conformational changes of M.HhaI upon binding DNA using smFRET. For the convenience of dye labelling, the Cys81 of M.HhaI was mutated to serine in global conformational studies. The other natural cysteines in M.HhaI were proven to be inert to the dye labelling. We doubly mutated selected sites on M.HhaI into cysteine and labelled them with donor (AF555) and acceptor (AF647) fluorophores in a random manner. We designed 13 mutants with FRET pairs distributed over all the domains of M.HhaI (Fig. S1^†), which are termed by the number of labelled sites as X-X (e.g., 90-210 means dyes are labelled at Q90C and K210C sites). The circular dichroism (CD) spectra showed that mutations and dye labelling did not disturb the structure of M.HhaI (Fig. S2^†). smFRET experiments were performed for the 13 mutants with M.HhaI in two conditions: (1) apo state, i.e., ∼40 pM M.HhaI in the T50 buffer (50 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, pH 7.5); and (2) DNA bound state, i.e., ∼40 pM M.HhaI with 2 μM SP_DNA (a specific DNA substrate) and 200 μM AdoMet in the T50 buffer.The experiments were conducted on a home-built inversed confocal fluorescence microscope.¹³ The ratio of the detected photon number in the acceptor channel over that in the acceptor and donor channels together was measured and is termed as apparent FRET efficiency (E_app). The E_app distribution histograms of the mutants in two conditions are presented in Fig. 1A and S3.^† The E_app peak at around 0.12 was caused by M.HhaI molecules labelled with only a bright donor (no acceptor or acceptor having been bleached), and was ignored in the analysis. The peaks at higher E_app were attributed to the effective FRET signal. All the mutants of M.HhaI in the apo state exhibited similar high E_app peaks, suggesting that apo M.HhaI stays in a collapsed conformation. After binding with DNA, lower E_app peaks appeared. To compare with the crystal structure, the true FRET efficiency (E) for mutants in the DNA bound state was calculated by rectifying the influence of crosstalk between the donor and acceptor channels, detection efficiency, and background photons in two channels. We plotted E of the DNA bound M.HhaI with the distance (d) provided by the crystal structure (Fig. 1B). The E–d curve showed clear anti-correlation and can be fitted well via a theoretical equation, suggesting that the DNA bound M.HhaI stays in a folded state characterized by the crystal structure. Urea denaturation experiments of 129-210 and 142-210 mutants supported these assignments. With the increase in the urea concentration, DNA bound M.HhaI showed the denaturation pattern of folded proteins, while apo M.HhaI showed the denaturation pattern of denatured proteins (Fig. S4^†).Open in a separate windowFig. 1(A) FRET histograms of several representative mutants in apo M.HhaI (gray) and in DNA bound M.HhaI (red) in the T50 buffer. (B) Corrected FRET efficiency of all the mutants in DNA bound state vs. the distance measured from the crystal structure (5MHT). Red line is the fitting curve obtained using the theoretical equation: E = R₀⁶/(R₀⁶ + (d + l)⁶), where R₀ = 51 Å is the Förster distance of the FRET pair of AF555 and AF647, and l = 12 Å is the equivalent length of linkers and dyes.This global conformational regulation upon DNA substrate binding was not expected from the crystal structure, nor has it been directly observed before. We also performed smFRET experiments on the binary complex of M.HhaI and AdoMet. AdoMet did not change the overall collapsed conformation of M.HhaI (Fig. S5^†). We speculated that the different appearance of apo M.HhaI in our smFRET and crystallographic studies was due to different experimental conditions. A high salt concentration will make the folded state more stable. During crystallization, M.HhaI as well as the salts in buffer were highly concentrated. To confirm this, we conducted smFRET experiments at high salt concentrations using the T1000 buffer (50 mM Tris–HCl, 1000 mM NaCl, 1 mM EDTA, pH 7.5). Indeed, new and lower E_app peaks appeared in apo M.HhaI (Fig. S6^†). At such high salt concentrations, the affinity between M.HhaI and DNA was too weak to form a complex. To compare apo M.HhaI with DNA bound M.HhaI in the T1000 buffer, we connected the 142-210 mutant (without C81S mutation) with the DNA substrate by a covalent bond. The 142-210 mutant was doubly labelled after it reacted with F-DNA, which is a derivative of SP_DNA with the hydrogen on carbon 5 of target cytosine substituted by fluorine in the presence of AdoMet. Due to the substitution of fluorine, the covalent bond between the Cys81 of M.HhaI and the carbon 6 of target cytosine could not be cleaved after methyl transfer and was used for the crystallographic study of the M.HhaI and DNA complex.^2,14 We termed the covalently bound complex of M.HhaI and DNA as 142-210_F-DNA^Me. As a control, we observed that in both the covalently bound and non-covalently bound complexes, M.HhaI exhibited the same folded conformation in the T50 buffer (Fig. S7^†). Then, in the T1000 buffer, we found the same E_app peaks for the apo 142-210 mutant and the covalently bound complex of M.HhaI and DNA (both 142-210_F-DNA^Me and 142-210_F-DNA^Me·AdoHcy) (Fig. 2A), indicating that at a high salt concentration apo M.HhaI indeed adopts the structure as that determined by X-ray crystallography. The results on other mutants reached the same conclusion. Since our experimental condition of low salt is closer to the real physiological condition, we believe that our observation more realistically reflects the status of M.HhaI in vivo.Open in a separate windowFig. 2(A) FRET histograms of apo 142-210 (gray), 142-210_F-DNA^Me (red) and 142-210_F-DNA^Me with AdoHcy (blue) in the T1000 buffer. (B) FRET histograms of apo 142-210 at different concentrations of NaCl. Tn means that in the Tris buffer the concentration of NaCl is n mM. (C) Single-molecule fluorescence trace of apo 142-210 (upper panel) and corresponding E_app trace (lower panel) in T50 buffer. (D) The calculated kinetic parameters k₊ (from unfolded to prefolded, red) and k₋ (from prefolded to unfolded, black) for the transition between unfolded and prefolded states of apo M.HhaI at different salt concentrations.Careful scrutinization of the smFRET histogram revealed that apo M.HhaI in the T50 buffer has two FRET components. We assigned the species with lower E_app as the prefolded state (the tertiary structure is partially maintained) because it behaves more or less like a folded protein but is much less stable than the M.HhaI–DNA complex under urea denaturation (Fig. S8^†). The one with higher E_app was assigned as the unfolded state (the tertiary structure is mostly destroyed) because it behaves as a typical denatured protein under urea denaturation (Fig. S4^†). Due to partially maintaining the tertiary structure, the prefolded state is more extended than the unfolded state such that its E_app is lower. As shown in Fig. 2B, with the increase in salt concentration, the proportion of the prefolded state increased and its E_app peak shifted toward that of 142-210_F-DNA^Me in the T1000 buffer (Fig. S9^†), suggesting that the partially folded prefolded state of apo M.HhaI under low salt concentration converted into a fully folded state at high salt concentration because a high salt concentration stabilizes the folded crystal structure.To capture the transition between the two states, we tracked the smFRET trace on the surface immobilized apo 142-210 mutant under a total internal reflection fluorescence microscope (TIRFM).¹⁵ Photon traces of donor and acceptor were recorded individually for each molecule (Fig. 2C, upper panel), from which E_app–time trace was calculated (Fig. 2C, lower panel). We then quantified the transition rates between the unfolded and prefolded states of apo M.HhaI from T0 (50 mM Tris–HCl, 1 mM EDTA, pH 7.5) to T1000 buffers by statistical analysis on the fluorescence traces. Transition from the unfolded state to prefolded state was designated as the forward reaction. As the results show in Fig. 2D, with the increase in the salt concentration, k₊ increases significantly while k₋ hardly changes. Overall, the global conformational transition of M.HhaI happens at the time scale of several hundred milliseconds. It is possible that M.HhaI prefers to bind DNA substrates in the prefolded state, from which it is easier to adjust into the folded conformation compared to starting from the unfolded state. The transition between the prefolded and unfolded states may serve as an additional regulation step for the substrate binding process.Thus, by examining the global conformational dynamics of M.HhaI, we observed three conformational states of M.HhaI, i.e., the unfolded state and prefolded state for apo M.HhaI (predominant in the physiological salt concentration), and the folded state for M.HhaI, which exists in the complex with DNA and apo M.HhaI at a high salt concentration. The conformational transition of apo M.HhaI and the conformational reorganization upon DNA binding on the global scale may play a critical role in the substrate recognition process.Next, we focused on the conformational changes of the catalytic loop during the catalytic process. First, we performed confocal-based smFRET experiments on the 90-210 mutant. In this study, we reserved Cys81 by blocking its accessibility with a DNA substrate during dye labelling (see the ESI^† for details). To learn about the pre-catalytic steps, smFRET measurements were performed in the following four situations: (1) M.HhaI with SP_DNA, (2) M.HhaI with SP_DNA in the presence of AdoHcy, (3) M.HhaI with NS_DNA (nonspecific DNA substrate), and (4) M.HhaI with NS_DNA in the presence of AdoHcy. For the former two situations, the experiments were conducted in the T50 buffer. To improve the affinity between M.HhaI and NS_DNA, the experiments for the last two situations were conducted in the T0 buffer. The results showed that when the substrate is SP_DNA, the catalytic loop stays open (E_app = 0.54, calculated d = 50 Å) in the binary complex of M.HhaI and SP_DNA. The loop then closes (E_app = 0.84, calculated d = 37 Å) after forming the ternary complex of M.HhaI, SP_DNA and AdoHcy (Fig. 3A). When the substrate is NS_DNA, the loop always stays open whether there is AdoHcy or not (Fig. 3B). To the best of our knowledge, the conformations of catalytic loop in the first and last two situations have not been directly observed before. Our results directly showed that the catalytic loop stays open during the target searching process and only closes when both the specific substrate and cofactor exist. This observation is essential to fully elucidate the mechanism of catalytic DNA methylation.Open in a separate windowFig. 3(A) FRET histograms of 90-210 (gray) that bind with specific DNA (labelled as SP_DNA) in the presence (blue) and absence of AdoHcy (red). (B) FRET histograms of 90-210 (gray), that bind with nonspecific DNA (labelled as NS_DNA) in the presence (blue) and absence of AdoHcy (red). (C) FRET histograms of binary complex of 90-210_F-DNA^Me (green) and ternary complex of 90-210_F-DNA^Me·AdoHcy (blue). 90-210 binding with nonspecific DNA is displayed (dashed line) as reference for the open state of loop. (D) The second order cross correlation curve G_AD of 90-210 (black) in the T1000 buffer and corresponding fitting curve (blue line). The G_AD of 142-210 is shown as control (red).To learn the post-catalytic steps, we detected the conformational changes of the loop during product release using F-DNA. The substitution of hydrogen for fluorine in the target cytosine pauses the reaction at the step right after methyl transfer, making the system with F-DNA a good model to study the post-catalytic step. smFRET measurements were conducted on the complex of 90-210_F-DNA^Me with and without AdoHcy. In the ternary complex with AdoHcy, the catalytic loop stays in a fully closed state (E_app = 0.82), while the loop stays in a half-open state (E_app = 0.72) in the binary complex 90-210_F-DNA^Me (without a cofactor) (Fig. 3C). To find out whether the single FRET peak represents a single half-open state or is due to the fast interchange between the open and closed states, we conducted the FRET-FCS experiment on a home-built scanning confocal microscope.¹⁶ No dynamic relaxation process was observed in 90-210_F-DNA^Me (Fig. S10^†), confirming that the FRET peak signals a single half-open state. This should be an important intermediate during the product release process that has not be recognized before. It suggests that immediately after the release of the product cofactor AdoHcy, the catalytic loop will spontaneously move out toward the open state and be ready to release the methylated base.In previous kinetic studies, the rate of catalytic loop flipping was concealed by the base flipping such that the true loop flipping rates can only be estimated.^5,6 Here, we were able to directly detect the dynamics of the catalytic loop by using the FRET-FCS technique with and without the substrate under equilibrium conditions. We measured the relaxation rate of the loop motion of apo M.HhaI in the T1000 buffer, where we had proven that apo M.HhaI stays in a folded state. Though the loop of apo M.HhaI showed a similar E_app peak to that of 90-210_F-DNA^Me in the T1000 buffer (Fig. S11^†), they have very different dynamic behaviours. A clear anti-correlation was detected in the cross correlation curve (G_AD) for apo 90-210, proving the existence of dynamic motion of the catalytic loop (Fig. 3D). As a control, the anti-correlation in the 142-210 mutant was much weaker, which may indicate that when the catalytic loop flips, other parts of M.HhaI make slight conformational adjustment as well. By including the recently developed third-order FRET-FCS calculation,¹⁷ we determined that k_open = (5.3 ± 0.3) × 10³ s⁻¹ and k_close = (1.6 ± 0.1) × 10³ s⁻¹. These values are faster than the rates estimated before^5,6 but land in the region suggested by the NMR studies on global dynamic motions.⁸ The E_app corresponding to the loop in closed and open states were calculated to be E^closed_app = 0.77 ± 0.09 and E^open_app = 0.44 ± 0.07. Because the smFRET experiment was performed on free molecules in solution, while the FRET-FCS experiment was performed on immobilized molecules on surface with different apparatuses, E_app has a systematic deviation. After correction, the true FRET efficiencies are consistent with the assignment on the loop open and closed states in the smFRET measurement, suggesting that the catalytic loop in apo M.HhaI swings in a broad range. The intrinsically high dynamic behaviour of the loop enables it to react quickly to the target recognition and base flipping and to reorganize concertedly with these processes. In contrast, on binding with DNA, the motion of loop is restrained to a smaller scale and exhibited in the binary complex of M.HhaI and SP_DNA as well as in the ternary complex of M.HhaI, SP_DNA and AdoHcy, where weaker anti-correlation was observed (Fig. S12^†). Their calculated E_app from the second- and third-order FCS curves confirmed that the motion represents local fluctuation around the potential minima of the open and closed states, respectively.In summary, we have used single-molecule fluorescence techniques, smFRET and FCS, to study the conformational dynamics of M.HhaI on two scales. On the global scale, we found that under the physiological salt concentration, apo M.HhaI does not possess the structure characterized by X-ray crystallography; instead, it interchanges between unfolded and prefolded states. It undergoes global conformational reorganizations to form a fully folded structure after the substrate recognition. On the local scale, we observed the conformational changes of catalytic loop during the catalytic process and found that the loop closes only when both the correct substrate and the cofactor are in correct positions. The rates of the loop flipping were directly quantified. These results provided new insights into the dynamic mechanisms of the catalytic DNA methylation. In addition, our results emphasized the fact that certain proteins in aqueous solutions may adopt conformations different from their crystal structures. Therefore, the crystal data should be cautiously used.     

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43

The N-terminal domain of the RNA binding protein TDP-43 (NTD) is essential to both physiology and proteinopathy; however, elucidation of its folding/unfolding still remains a major quest. In this study, we have investigated the biophysical behavior of intermediate ensembles employing all-atom molecular dynamics simulations in 8 M urea accelerated with high temperatures to achieve unfolded states in a confined computation time. The cumulative results of the 2.75 μs simulations show that unfolding of the NTD at 350 K evolves through different stable and meta-stable intermediate states. The free-energy landscape reveals two meta-stable intermediates (I_N and I_U) stabilized by non-native interactions, which are largely hydrophilic and highly energetically frustrated. A single buried tryptophan residue, W80, undergoes solvent exposure to different extents during unfolding; this suggests a structurally heterogeneous population of intermediate ensembles. Furthermore, the structure properties of the I_N state show a resemblance to the molten globule (MG) state with most of the secondary structures intact. The unfolding of the NTD is initiated by the loss of β-strands, and the unfolded (U) states exhibit a population of non-native α-helices. These non-native unfolded intermediate ensembles may mediate protein oligomerization, leading to the formation of pathological, irreversible aggregates, characteristics of disease pathogenesis.

The N-terminal domain of the RNA binding protein TDP-43 (NTD) is essential to both physiology and proteinopathy; however, elucidation of its folding/unfolding still remains a major quest.     

The effects of osmolytes on in vitro kinesin-microtubule motility assays

The gliding motility of microtubule filaments has been used to study the biophysical properties of kinesin motors, as well as being used in a variety of nanotechnological applications. While microtubules are generally stabilized in vitro with paclitaxel (Taxol®), osmolytes such as polyethylene glycol (PEG) and trimethylamine N-oxide (TMAO) are also able to inhibit depolymerization over extended periods of time. High concentrations of TMAO have also been reported to reversibly inhibit kinesin motility of paclitaxel-stabilized microtubules. Here, we examined the effects of the osmolytes PEG, TMAO, and glycerol on stabilizing microtubules during gliding motility on kinesin-coated substrates. As previously observed, microtubule depolymerization was inhibited in a concentration dependent manner by the addition of the different osmolytes. Kinesin-driven motility also exhibited concentration dependent effects with the addition of the osmolytes, specifically reducing the velocity, increasing rates of pinning, and altering trajectories of the microtubules. These data suggest that there is a delicate balance between the ability of osmolytes to stabilize microtubules without inhibiting motility. Overall, these findings provide a more comprehensive understanding of how osmolytes affect the dynamics of microtubules and kinesin motors, and their interactions in crowded environments.

Kinesin-driven motility was shown to be adversely affected in a concentration dependent manner by the addition of osmolytes: glycerol, polyethylene glycol, and trimethylamine N-oxide.     

The role of urea in the solubility of cellulose in aqueous quaternary ammonium hydroxide

We examine the role of water and urea in cellulose solubility in tetrabutylammonium hydroxide (TBAH). Molecular dynamics simulations were performed for several different solvent compositions with a fixed cellulose fraction. For each composition, two simulations were carried out with cellulose fixed in each of the crystalline and the dissolved states. From the enthalpy and the entropy of the two states, the difference in Gibbs free energy (ΔG) and hence the spontaneity is determined. A comparison with solubility experiments showed a strong correlation between the calculated ΔG and the experimental measurements. A breakdown of the enthalpic and entropic contributions reveals the roles of water and urea in solubility. At high water concentration, a drop in solubility is attributed to both increased enthalpy and decreased entropy of dissolution. Water displaces strong IL–cellulose interactions for weaker water–cellulose interactions, resulting in an overall enthalpy increase. This is accompanied by a strong decrease in entropy, which is primarily attributed to both water and the entropy of mixing. Adding urea to TBAH(aq) increases solubility by an addition to the mixing term and by reducing losses in solvent entropy upon dissolution. In the absence of urea, the flexible [TBA]⁺ ions lose substantial degrees of freedom when they interact with cellulose. When urea is present, it partially replaces [TBA]⁺ and to a lesser extent OH⁻ near cellulose, losing less entropy because of its rigid structure. This suggests that one way to boost the dissolving power of an ionic liquid is to limit the number of degrees of freedom from the outset.

We examine the role of water and urea in cellulose solubility in tetrabutylammonium hydroxide (TBAH).     

Anionic effects on the structure and dynamics of water in superconcentrated aqueous electrolytes

Dissolved ions in aqueous solutions are ubiquitous in a variety of systems and the addition of ions to water gives rise to dramatic effects on the properties of water. Due to a significant role of ions in the structure and dynamics of water, the ionic conditions, such as the ion type and concentration, have been considered as critical factors. Here we study the effects of anions on the structure and dynamics of water in aqueous electrolytes for various lithium salt concentrations via extensive molecular dynamics simulations. Our results demonstrate that a certain amount of salt is needed to show the different properties of water caused by the presence of different types of anion. Below the cutoff concentration, most features of water show the same characteristics in spite of the presence of different anions. In the superconcentrated limit, we find that full disruption of the hydrogen bond network between water molecules occurs for most anions investigated, indicating that the effect of the water–water interaction becomes negligible. However, a certain type of anion could enhance an ion-pairing of cations and anions and the water–water interaction remains considerable even in the superconcentrated limit. We further investigate the cationic and anionic hydration shell structures and dynamics, revealing their dependence on the anion type and the salt concentration. Finally, we observe that the anionic effects on water extend to the dynamics of water molecules, such as an anionic dependence of the onset of subdiffusive translation and anisotropic rotation.

The effects of anions on the properties of water are examined for various salt concentrations.     

Insight into structural properties of polyethylene glycol monolaurate in water and alcohols from molecular dynamics studies

By means of molecular dynamics (MD) simulations, we explored the structural properties of polyethylene glycol monolaurate (PEGML) in water and in various aliphatic alcohols (methanol, ethanol, 2-propanol, 2-butanol, tert-butanol, and 1-pentanol). The PEGML and the alcohols were simulated using the optimized potentials for liquid simulations, all-atom (OPLS-AA) force field and water using the extended simple point charge (SPC/E) model. From the isothermal-isobaric (NPT, constant number of particles, constant pressure, and constant temperature) ensemble, we extracted the densities from the simulations and compared them with those from experimental results in order to confirm the validity of the selected force fields. The densities from MD simulations are in good agreement with the experimental values. To gain more insight into the nature of interactions between the PEGML and the solvent molecules, we analyzed the hydrogen-bonds, the electrostatic (Coulomb) interactions, and the van der Waals (Lennard-Jones) interaction energies extracted from MD simulations. The results were further strengthened by computing the solvation free energy by employing the free energy perturbation (FEP) approach. In this method, the free energy difference was computed by using the Bennet Acceptance Ratio (BAR) method. Moreover, the radial distribution functions were analyzed in order to gain more understanding of the solution behavior at the molecular level.

By means of molecular dynamics (MD) simulations, we explored the structural properties of polyethylene glycol monolaurate (PEGML) in water and in various aliphatic alcohols (methanol, ethanol, 2-propanol, 2-butanol, tert-butanol, and 1-pentanol).     

Exploring interfacial dynamics in homodimeric S-ribosylhomocysteine lyase (LuxS) from Vibrio cholerae through molecular dynamics simulations

To the best of our knowledge, this is the first molecular dynamics simulation study on the dimeric form of the LuxS enzyme from Vibrio cholerae to evaluate its structural and dynamical properties including the dynamics of the interface formed by the two monomeric chains of the enzyme. The dynamics of the interfacial region were investigated in terms of inter-residual contacts and the associated interface area of the enzyme in its ligand-free and ligand–bound states which produced characteristics contrast in the interfacial dynamics. Moreover, the binding patterns of the two inhibitors (RHC and KRI) to the enzyme forming two different enzyme–ligand complexes were analyzed which pointed towards a varying inhibition potential of the inhibitors as also revealed by the free energies of ligand binding. It is shown that KRI is a more potent inhibitor than RHC – a substrate analogue, showing correlation with experimental data. Moreover, the role of a loop in chain B of the enzyme was found to facilitate the binding of RHC similar to that of the substrate, while KRI demonstrates a differing binding pattern. The computation of the free energy of binding for the two ligands was also carried out via thermodynamic integration which ultimately served to correlate the dynamical properties with the inhibition potential of two different ligands against the enzyme. Furthermore, this successful study provides a rational to suggest novel LuxS inhibitors which could become promising candidates to treat the diseases caused by a broad variety of bacterial species.

To the best of our knowledge, this is the first molecular dynamics simulation study on the dimeric form of the LuxS enzyme from Vibrio cholerae to evaluate its structural and dynamical properties including the dynamics of the interface formed by the two monomeric chains of the enzyme.     

Anomalous dynamics of water at the octopeptide lanreotide surface

This work reports the study of water dynamics close to the cyclic octapeptide lanreotide from atomistic simulations of hydrated lanreotide, a cyclic octapeptide. Calculation of the hydrogen bonds between water molecules allows mapping of the hydrophilic regions of lanreotide. Whereas a super-diffusivity of the interfacial water molecules is established, a slowdown in rotational dynamics is observed, involving a decoupling between both processes. Acceleration in translation dynamics is connected to the hopping process between hydrophilic zones. Microscopically, this is correlated with the weakness of the interfacial hydrogen bonding network due to a hydrophobic interface at the origin of the interfacial sliding of water molecules. Heterogeneous rotational dynamics of water molecules close the lanreotide surface is evidenced and connected to heterogeneous hydration.

Molecular dynamics simulations of a hydrated mutated lanreotide, a cyclic octapeptide, were carried out to characterize its hydration state. We studied the water dynamics close to the peptide using atomistic simulations.     

T54R mutation destabilizes the dimer of superoxide dismutase 1T54R by inducing steric clashes at the dimer interface

Mutations cause abnormalities in protein structure, function and oligomerization. Different mutations in the superoxide dismutase 1 (SOD1) protein cause its misfolding, loss of dimerization and aggravate its aggregation in the amyotrophic lateral sclerosis disease. In this study, we report the mechanistic details of how a threonine-to-arginine mutation at the 54^th position (T54R) of SOD1 results in destabilization of the dimer interface of SOD1^T54R. Using computational and experimental methods, we show that the T54R mutation increases fluctuation of the mutation-harboring loop (R54-loop) of SOD1^T54R. Fluctuation of this loop causes steric clashes that involve arginine-54 (R54) and other residues of SOD1^T54R, resulting in loss of inter-subunit contacts at the dimer interface. Since the T54 residue-containing loop is necessary for the dimerization of wild-type SOD1, fluctuation of the R54-loop, steric clashes involving R54 and loss of inter-subunit contacts give rise to the loss of SOD1^T54R dimer stability. This correlates to energetically unfavorable tethering of the monomers of SOD1^T54R. The outcome is gradual splitting of SOD1^T54R dimers into monomers, thereby exposing the previously buried hydrophobic interface residues to the aqueous environment. This event finally leads to aggregation of SOD1^T54R. T54R mutation has no effect in altering the relative positions of copper and zinc ion binding residues of SOD1^T54R. The native SOD1 structure is stable, and there is no destabilizing effect at its dimer interface. Overall, our study reveals the intricate mechanism of T54R mutation-associated destabilization of the dimer of the SOD1^T54R protein.

T54R mutation destabilizes the dimer of SOD1^T54R.     

Ultrafast broadband nonlinear optical properties and excited-state dynamics of two bis-chalcone derivatives

The development of organic nonlinear optical (NLO) chromophores is vital for various fields such as two-photon biomedical imaging, optical limiting, etc. In this work, two bis-chalcone molecules 1,4-bis[3-(2,4-dimethoxyphenyl)-2-acryloyl]benzene (C1) and 4,4′-bis[3-(2,4-bimethoxy phenyl)-2-acryloyl]biphenyl (C2) were synthesized and characterized. The excited-state dynamics of these two chromophores were studied using femtosecond transient absorption (TA) measurements. And their broadband nonlinear absorption properties and optical limiting (OL) response were investigated by femtosecond open-aperture Z-scan and intensity-dependent transmittance measurements in the wavelength range from 515 nm to 800 nm, respectively. The TA results demonstrate that C2 has strong excited-state absorption behavior and longer lifetime. In addition, the nonlinear absorption response of C2 was found to be superior to that of C1 in the visible range after 500 nm, which is attributed to a two-photon-absorption induced excited-state absorption mechanism. These results indicate that the nonlinear optical response and excited-state dynamics in bis-chalcone compounds could be enhanced via intramolecular charge-transfer.

The introduction of a benzene ring largely affects the excited-state absorption spectra and dynamics of these chromophores.     

Proof of concept web application for understanding the energetic basis of oligonucleotide unfolding

Measuring and quantifying thermodynamic parameters that determine both the stability of and interactions between biological macromolecules are an essential and necessary complement to structural studies. Although basic thermodynamic parameters for an observed process can be readily obtained, the data interpretation is often slow and analysis quality can be extremely variable. We have started to develop a web application that will help users to perform thermodynamic characterizations of oligonucleotide unfolding. The application can perform global fitting of calorimetric and spectroscopic data, and uses a three-state equilibrium model to obtain thermodynamic parameters for each transition step – namely, the Gibbs energy, the enthalpy, and the heat capacity. In addition, the application can define the number of K⁺ ions and the number of water molecules being released or taken up during unfolding. To test our application, we used UV spectroscopy, circular dichroism, and differential scanning calorimetry to monitor folding and unfolding of a model 22-nucleotide-long sequence of a human 3′-telomeric overhang, known as Tel22. The obtained data were uploaded to the web application and the global fit revealed that unfolding of Tel22 involves at least one intermediate state, and that K⁺ ions are released during the unfolding, whereas water molecules are taken up.

A novel web application: performing global fitting of oligonucleotide unfolding experimental data in style.     

Probing intrinsic dynamics and conformational transition of HIV gp120 by molecular dynamics simulation

The HIV envelope glycoprotein gp120 has evolved two distinct conformational states to balance viral infection and immune escape. One is a closed state resistant to most neutralization antibodies, and the other is an open state responsible for the binding of the receptor and coreceptors. Although the structures of gp120 in these two conformational states have been determined, a detailed molecular mechanism involving intrinsic dynamics and conformational transition is still elusive. In this study, μs-scale molecular dynamics simulation is performed to probe molecular dynamics and conformational transition away from the open state and approach the closed state. Our results reveal that open gp120 shows a larger structural deviation, higher conformational flexibility, and more conformational diversity than the form in the closed state, providing a structural explanation for receptor or coreceptor affinity at the open state and the neutralization resistance of closed conformation. Seven regions with greatly decreased coupled motions in the open states have been observed by dynamic cross-correlation analysis, indicating that conformational transition can be mainly attributed to the relaxation of intrinsic dynamics. Three conformations characterized by the structural orientations of the V1/V2 region and the V3 loop, suggesting gp120 is intrinsically dynamic from the open state to the closed state. Taken together, these findings shed light on the understanding of the conformational control mechanism of HIV.

The HIV envelope glycoprotein gp120 has evolved two distinct conformational states to balance viral infection and immune escape.     

Egress and invasion machinery of malaria: an in-depth look into the structural and functional features of the flap dynamics of plasmepsin IX and X

Plasmepsins, a family of aspartic proteases expressed by Plasmodium falciparum parasite, have been identified as key mediators in the onset of lethal malaria. Precedence has been placed on this family of enzymes due their essential role in the virulence of the parasite, thus highlighting their importance as novel drug targets. A previously published study by our group proposed a set of parameters used to define the flap motion of aspartic proteases. These parameters were used in the study of Plm I–V and focused on the flap flexibility as well as structural dynamics. Recent studies have highlighted the essential role played by Plm IX and X in egress and invasion of the malarial parasite. This study aims to close the gap on the latter family, investigating the flap dynamics of Plms IX and X. Molecular dynamics simulations demonstrated an “open and close” mechanism at the region of the catalytic site. Further computation of the dihedral angles at the catalytic region revealed tractability at both the flap tip and flexible loop. This structural versatility enhances the interaction of variant ligand sizes, in comparison to other Plm family members. The results obtained from this study signify the essential role of structural flap dynamics and its resultant effect on the binding landscapes of Plm IX and X. We believe that this unique structural mechanism may be integrated in the design and development of effective anti-malarial drugs.

A molecular dynamic study of the infiltrating machinery of malaria, an in-depth look in the flap and loop dynamics of Plm IX and X.     

Membrane partition of bis-(3-hydroxy-4-pyridinonato) zinc(ii) complexes revealed by molecular dynamics simulations

The class of 3-hydroxy-4-pyridinone ligands is widely known and valuable for biomedical and pharmaceutical purposes. Their chelating properties towards biologically-relevant transition metal ions highlight their potential biomedical utility. A set of 3-hydroxy-4-pyridinone Zn(ii) complexes at different concentrations was studied for their ability to interact with lipid phases. We employed umbrella sampling simulations to attain the potential-of-mean force for a set of ligands and one Zn(ii) complex, as these permeated a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) hydrated bilayer system. In addition, we used conventional molecular dynamics simulations to study the behavior of various Zn(ii) complexes in hydrated bilayer systems. This work discusses: (i) the partition of 3-hydroxy-4-pyridinone ligands to bilayer phases; (ii) self-aggregation in crowded environments of Zn(ii) complexes; and (iii) possible mechanisms for the membrane translocation of Zn(ii) complexes. We observed distinct interactions for the studied complexes, and distinct membrane partition coefficients (K_mem) depending on the considered ligand. The more hydrophobic ligand, 1-hexyl-3-hydroxy-2-methyl-4(1H)-pyridinone, partitioned more favorably to lipid phases (at least two orders of magnitude higher K_mem when compared to the other ligands), and the corresponding Zn(ii) complex was also prone to self-aggregation when an increased concentration of the complex was employed. We also observed that the inclusion of a coordinated water molecule in the parameterization of the Zn(ii) coordination sphere, as proposed in the available crystallographic structure of the complex, decreased the partition coefficient and membrane permeability for the tested complex.

The membrane partition of hydroxypyridinones and of zinc complexes explored by molecular dynamics.     

Computational investigations of the binding mechanism of novel benzophenone imine inhibitors for the treatment of breast cancer

4-Hydroxytamoxifen (4-OHT), the most common hormone used for the treatment of breast cancer, is a selective estrogen receptor modulator (SERM) inhibitor that acts as an antagonist in breast tissue and a partial agonist in the endometrium. However, the detailed molecular mechanism of 4-OHT structure modification has not been well investigated to date. Herein, molecular docking, molecular dynamics simulations and free energy calculations were performed to explore the mechanisms of the molecular interactions between newly designed benzophenone imines (BIs) and the three forms apo, antagonist and agonist of the human estrogen receptor hERα. The proposed inhibitors were designed by replacing the triarylethylene estrogenic scaffold found in 4-OHT with Schiff base triarylimine derivatives. The antiestrogen scaffold i.e. the O-alkyl side chain in 4-OHT was developed by incorporating an alanine amino acid side chain functionality into the triarylimine scaffold. Docking results reveal that the newly designed BIs bind to the hydrophobic open pocket of the apo and antagonist hERα conformations with higher affinity as compared to the natural and synthetic estrogen estradiol (E2) and 4-OHT. The analysis of the molecular dynamics simulation results based on six different systems of the best docked BI (5c) with hERα receptors demonstrates stable interactions, and the complex undergoes fewer conformational fluctuations in the open apo/antagonist hERα receptors as compared to the case of the closed agonist. In addition, the calculated binding free energies indicate that the main factor that contributes to the stabilization of the receptor–inhibitor complexes is hydrophobic interactions. This study suggests that the development of these Schiff base derivatives may be worth exploring for the preparation of new 4-OHT analogues.

4-Hydroxytamoxifen (4-OHT), the most common hormone used for the treatment of breast cancer, is a selective estrogen receptor modulator (SERM) inhibitor that acts as an antagonist in breast tissue and a partial agonist in the endometrium; therefore, it increases the risk of uterine cancer while lowering the risk of breast cancer recurrence.     

Dispersion-induced structural preference in the ultrafast dynamics of diphenyl ether

Dispersion interactions are omnipresent in large aromatic systems and influence the dynamics as intermolecular forces. The structural preference induced by dispersion interactions is demonstrated to influence the excited state dynamics of diphenyl ether (DPE) using femtosecond time-resolved transient absorption (TA) associated with quantum chemical calculations. The experimental results in aprotic solvents show that the S₁ state is populated upon irradiation at 267 nm with excess vibrational energy dissipating to solvent molecules in several picoseconds, and then decays via internal conversion (IC) within 50 ps as well as intersystem crossing (ISC) and fluorescence with a lifetime of nanoseconds. The polarity of the solvent disturbs the excited state energies and enhances the energy barriers of the ISC channel. Furthermore, the intermolecular dispersion interactions with protic solvents result in the OH–π isomer dominating in methanol and the OH–O isomer is slightly preferred in t-butanol in the ground state. The hydrogen bonded isomer measurements show an additional change from OH–O to OH–π geometry in the first 1 ps besides the relaxation processes in aprotic solvents. The time constants measured in the TA spectra suggest that the OH–O isomer facilitates IC. The results show that the OH–π isomer has a more rigid structure and a higher barrier for IC, making it harder to reach the geometric conical intersection through conformer rearrangement. This work enables us to have a good knowledge of how the structural preference induced by dispersion interactions affects excited state dynamics of the heteroaromatic compounds.

Dispersion interactions are omnipresent in large aromatic systems and influence the dynamics as intermolecular forces.     

Formation and dissociation of synthetic hetero-double-helix complex in aqueous solutions: significant effect of water content on dynamics of structural change

A 1 : 1 mixture of the ethynylhelicene pseudoenantiomers (M)-tetramer and (P)-pentamer, which possess hydrophilic terminal tri(ethyleneglycol) (TEG) groups, changes their structures in the water–THF (10 μM) solvent system between dissociated random-coils and an associated hetero-double-helix upon heating and cooling. A small change in water content between 30 and 33% significantly affects the dynamics of structural changes. At 30% water content, heating to 60 °C causes rapid formation of random-coil and cooling to 10 °C causes the rapid formation of hetero-double-helix, accompanied by repeated changes in Δε at 369 nm between 0 and −2000 cm⁻¹ M⁻¹. Heating and cooling experiments at constant rates between 60 and 10 °C resulted in sigmoidal curves in Δε/temperature profiles, which indicate rapid structural changes. Different phenomena occurred at 33% water content. Heating to 60 °C and cooling to 0 °C initially induced changes in Δε between 0 and −2000 cm⁻¹ M⁻¹, and repeated cycles gradually reduced the range between 0 and −500 cm⁻¹ M⁻¹. Heating and cooling experiments at constant rates between 60 and 10 °C caused small changes in Δε, and repeated cycles at 10 °C gradually increased Δε to −500 cm⁻¹ M⁻¹. These phenomena involved rapid changes in molecular structure and slow structural changes in the water–THF solvent system. The sharp switching of the dynamics of structural changes at water content between 30 and 33% indicated discontinuous structural changes in the hydration of TEG and/or in water clusters in the vicinity of oligomer molecules.

Significant structural changes by small change in water content from 30 to 33%.     

Experimental and molecular dynamics study into the surfactant effect upon coal wettability

In order to improve the wettability and permeability of coal seams, the water injection efficiency of coal seams has to be boosted, the amount of dust generation has to be reduced, and coal and gas outburst must be prevented, and a surfactant is used to modulate the coal surface wettability. In this work, taking coal samples from Pingdingshan mine in Henan as the research object, their surface chemistry was initially scrutinized and then coal surface engineering via surfactants was inspected by a contact angle test. The coal wettability was ameliorated with surfactants, particularly using the 1 wt% non-ionic surfactant Triton X-100, which elicited a 47% lower contact angle than the raw coal. The surface free energy of the coal sample modified by 1.0 wt% Triton X-100 was increased from 44.51 mN m⁻¹ to 49.52 mN m⁻¹. The microstructural characteristics of coal samples allowed leveraging the Wiser model to construct three kinds of surfactant-coal adsorption models to dissect the adsorption configuration of the system. The results indicate that the addition of surfactants increases both the interaction of water with the coal and the diffusion coefficient of water molecules, resulting in the coal surface transformation from hydrophobicity to hydrophilicity. Our current work can provide salutary guidance and reference for coal water injection and dust suppression.

The experimental and molecular dynamics studies show that the effective wettability of Triton X-100 is controlled by the strong π–π adsorption between hydrophobic end and coal molecule, and the hydrogen bond between hydrophilic end and water.     

The relative stability of trpzip1 and its mutants determined by computation and experiment

Six mutants of the tryptophan zipper peptide trpzip1 have been computationally and experimentally characterized. We determine the varying roles in secondary structure stability of specific residues through a mutation assay. Four of the mutations directly effect the Trp–Trp interactions and two of the mutations target the salt bridge between Glu5 and Lys8. CD spectra and thermal unfolding are used to determine the secondary structure and stability of the mutants compared to the wildtype peptide. Adaptive steered molecular dynamics has been used to obtain the energetics of the unfolding pathways of the mutations. The hydrogen bonding patterns and side-chain interactions over the course of unfolding have also been calculated and compared to wildtype trpzip1. The key finding from this work is the importance of a stabilizing non-native salt bridge pair present in the K8L mutation.

The single-point mutations of tprzip1 are indicated at left, and their relative energetics are compared at right.