Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

Isomerization Pathways of a Mismatched Base Pair of A:8OG in Free Duplex DNA.

The A:8OG base pair (bp) is the outcome of DNA replication of the mismatched C:8OG bp. A high A:8OG bp population increases the C/G to A/T transversion mutation, which is responsible for various diseases. MutY is an important enzyme in the error-proof cycle and reverts A:8OG to C:8OG bp by cleaving adenine from the A:8OG bp. Several X-ray crystallography studies have determined the structure of MutY during the lesion scanning and lesion recognition stages. Interestingly, glycosidic bond (χ) angles of A:8OG bp in those two lesion recognition structures were found to differ, which implies that χ-torsion isomerization should occur during the lesion recognition process. In this study, as a first step to understanding this isomerization process, we characterized the intrinsic dynamic features of A:8OG in free DNAs by a free energy landscape simulation at the all-atom level. In this study, four isomerization states were assigned in the order of abundance: Aanti:8OGsyn > Aanti:8OGanti > Asyn:8OGanti ≈ Asyn:8OGsyn. Of these bp states, only 8OG in Asyn:8OGanti was located in the extrahelical space, whereas the purine bases (A and 8OG) in the other bp states remained inside the DNA helix. Also, free energy landscapes showed that the isomerization processes connecting these four bp states proceeded mostly in the intrahelical space via successive single glycosidic bond rotations of either A or 8OG.

Computational Probing of the Folding Mechanism of Human Telomeric G-Quadruplex DNA.

The human telomeric (htel) sequences in the terminal regions of human telomeres form diverse G-quadruplex (GQ) structures. Despite much experimental efforts to elucidate the folding pathways of htel GQ, no comprehensive model of htel GQ folding has been presented. Here, we describe folding pathways of the htel GQ determined by state-of-the-art enhanced sampling molecular dynamics simulation at the all-atom level. Briefly, GQ folding is initiated by the formation of a single-hairpin and then followed by the formation of double-hairpins, which then branch via distinct folding pathways to produce different GQ topologies (antiparallel chair, antiparallel basket, hybrids 1 and 2, and parallel propeller). In addition to these double-hairpin states, three-triad and two-tetrad structures in antiparallel backbone alignment serve as key intermediates that connect the GQ folding and transition between two different GQs.

Complete photodissociation dynamics of CF2I2 in solution.

Photodissociation dynamics of CF2I2 in cyclohexane were evaluated by probing the C-F stretching mode over a wide time range after ultraviolet excitation using femtosecond infrared spectroscopy. After the ultrafast (<0.2 ps) state-selective photodissociation of CF2I2 as in the gas phase (267 nm excitation led to exclusive three-body dissociation (CF2 + I + I), 350 nm to exclusive two-body dissociation (CF2I + I), and 310 nm to a mixture of three- and two-body dissociations), various secondary reactions were observed. Once produced, some nascent CF2 radicals immediately formed a complex with the departing I atom (ICF2), which produced either CF2I or CF2 radicals. The produced CF2I geminately recombined with the I atom, whereas the CF2 radical reacted bimolecularly to produce C2F4 with a diffusion-limited rate constant of 8.1 × 109 M-1 s-1. Some nascent CF2I radicals were produced with sufficient excess energy to further dissociate into CF2 and I, or immediately reacted with the dissociated I atom to form the I2-CF2 isomer that rapidly dissociated into CF2 and I2. Other nascent CF2I radicals geminately recombined with the I atom with various time constants. Thus, the nascent photoproducts, CF2 and CF2I take various reaction paths: complex formation, secondary dissociation, isomer formation, and fast and slow germinate rebindings. The ensuing reaction path of the nascent photoproduct is dictated by its internal energy as well as solvent environment, which leads to different interactions between the photoproduct and solvent. Measurement over a broad time range with a structure-sensitive probe could reveal the fate of all the reaction intermediates, which allows evaluation of the complete reaction dynamics in solution.

Insilico direct folding of thrombin-binding aptamer G-quadruplex at all-atom level.

The reversible folding of the thrombin-binding DNA aptamer G-quadruplexes (GQs) (TBA-15) starting from fully unfolded states was demonstrated using a prolonged time scale (10-12 µs) parallel tempering metadynamics (PTMetaD) simulation method in conjunction with a modified version of the AMBER bsc1 force field. For unbiased descriptions of the folding free energy landscape of TBA-15, this force field was minimally modified. From this direct folding simulation using the modified bsc1 force field, reasonably converged free energy landscapes were obtained in K+-rich aqueous solution (150 mM), providing detailed atomistic pictures of GQ folding mechanisms for TBA-15. This study found that the TBA folding occurred via multiple folding pathways with two major free energy barriers of 13 and 15 kcal/mol in the presence of several intermediate states of G-triplex variants. The early formation of these intermediates was associated with a single K+ ion capturing. Interestingly, these intermediate states appear to undergo facile transitions among themselves through relatively small energy barriers.

Free energy landscape and transition pathways from Watson-Crick to Hoogsteen base pairing in free duplex DNA.

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson-Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine-thymine (A-T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events.

Rebinding dynamics of NO to microperoxidase-8 probed by time-resolved vibrational spectroscopy.

Femtosecond vibrational spectroscopy was used to probe the rebinding kinetics of NO to microperoxidase-8 (Mp), an ideal model system for the active site of ligand-binding heme proteins, including myoglobin and hemoglobin, after the photodeligation of MpNO in glycerol/water (G/W) solutions at 294 K. The geminate rebinding (GR) of NO to Mp in viscous solutions was highly efficient and ultrafast and negligibly dependent on the solution viscosity, which was adjusted by changing the glycerol content from 65% to 90% by volume in G/W mixtures. The kinetics of the GR of NO to Mp in viscous solutions was well represented by an exponential function with a time constant of ca. 11 ps. Although the kinetic traces of the GR of NO to Mp in solutions with three different viscosities (18, 81, and 252 cP) almost overlap, they show a slight difference early in the decay process. The kinetic traces were also described by the diffusion-controlled reaction theory with a Coulomb potential. Since the ligand is deligated in a neutral form, an ionic pair of NO(-) and Mp(+) may be produced before forming the Mp-NO bond by an electron transfer from Mp to NO as the deligated NO is sufficiently near to the Fe atom of Mp. The strong reactivity between NO and ferrous heme may arise from the Coulomb interaction between the reacting pair, which is consistent with the harpooning mechanism for NO binding to heme.

Three-State Diffusion Model of DNA Glycosylase Translocation along Stretched DNA as Revealed by Free Energy Landscapes at the All-Atom Level.

DNA glycosylases play key roles in the maintenance of genomic integrity. These enzymes effectively find rare damaged sites in DNA and participate in subsequent base excision repair. Single-molecule and ensemble experiments have revealed key aspects of this damage-site searching mechanism and the involvement of facilitated diffusion. In this study, we describe free energy landscapes of enzyme translocation along nonspecific DNA obtained using a fully atomistic molecular dynamics (MD) simulation of a well-known DNA glycosylase, human 8-oxoguanine DNA glycosylase 1 (hOGG1). Based on an analysis of simulated free energy profiles, we propose a three-state model for the damage-site searching mechanism. In the three states, named the L1, L2, and L3 states, the L1 state is a helical sliding mode in close contact with DNA, whereas the L2 state is a major- or minor-groove tracking mode in loose contact with DNA and the L3 state is a two-dimensional freely diffusing mode during which hOGG1 is somewhat removed from the DNA surface (∼24 Šaway from the surface). This three-state model well describes key experimental findings obtained from single-molecule and ensemble experiments and provides a unified molecular picture of the DNA lesion-searching mechanism of hOGG1.

Large tunneling effect on the hydrogen transfer in bis(µ-oxo)dicopper enzyme: a theoretical study.

Type-III copper-containing enzymes have dicopper centers in their active sites and exhibit a novel capacity for activating aliphatic C-H bonds in various substrates by taking molecular oxygen. Dicopper enzyme models developed by Tolman and co-workers reveal exceptionally large kinetic isotope effects (KIEs) for the hydrogen transfer process, indicating a significant tunneling effect. In this work, we demonstrate that variational transition state theory allows accurate prediction of the KIEs and Arrhenius parameters for such model systems. This includes multidimensional tunneling based on state-of-the-art quantum-mechanical calculations of the minimum-energy path (MEP). The computational model of bis(µ-oxo)dicopper enzyme consists of 70 atoms, resulting in a 204-dimensional potential energy surface. The calculated values of E(a)(H) - E(a)(D), A(H)/A(D), and the KIE at 233 K are -1.86 kcal/mol, 0.51, and 28.1, respectively, for the isopropyl ligand system. These values agree very well with experimental values within the limits of experimental error. For the representative tunneling path (RTP) at 233 K, the pre- and post-tunneling configurations are 3.3 kcal/mol below the adiabatic energy maximum, where the hydrogen travels 0.54 Å by tunneling. We found that tunneling is very efficient for hydrogen transfer and that the RTP is very different from the MEP. It is mainly heavy atoms that move as the reaction proceeds from the reactant complex to the pretunneling configuration, and the hydrogen atom suddenly hops at that point.

Improving All-Atom Force Field to Accurately Describe DNA G-Quadruplex Loops.

The DNA G-quadruplex (GQ) displays structural polymorphisms, and interactions between its loops and flanking sequences critically determine which of the diverse GQ conformers is adopted. All-atom molecular dynamics (MD) simulations of GQs are computationally challenging due to slow folding times and force field (ff) artifacts. In an earlier study, a direct folding simulation of the simplest DNA GQ (TBA15) was first reported using a modified version of the AMBER bsc1 ff (bsc1_vdW ff). Despite this successful folding simulation, it was later found that the bsc1_vdW ff is somewhat limited in terms of describing loop structures of GQs, which is problematic because GQ loop regions play key roles in ligand binding to modulate GQ activities. In this study, we further modified the bsc1_vdW ff to enhance the GQ loop prediction by fine-tuning a limited number of van der Waals (vdW) parameters of the standard AMBER bsc1 ff to improve the GQ loop distribution of a target GQ system (three-layered antiparallel GQ; mHtel21). Test simulations of this newly generated ff (bsc1_vdWL ff) on DNA GQs with diverse topologies (hybrid1, hybrid2, and parallel propeller) revealed that loop structures were predicted more accurately than by the bsc1_vdW ff. We consider that enhanced sampling MD simulation methods in combination with bsc1_vdWL provide useful simulation protocols for resolving outstanding issues of DNA GQ folding and GQ/ligand binding at the all-atom level.

Multiple stepwise pattern for potential of mean force in unfolding the thrombin binding aptamer in complex with Sr2+.

Using all-atom molecular dynamics simulation in conjunction with umbrella sampling, we obtained the unfolding free energy and the force extension profiles of the thrombin binding DNA aptamer (15-TBA) in complex with Sr(2+) (Protein Data Bank code: 1RDE). The resulting potential of mean force (PMF) displays a multiple stepwise pattern with distinct plateau regions. The detailed analysis of the simulation result indicated that each plateau was created by the interplay of the metal ion interacting with self-arranging guanine bases and the successive uptakes of water molecules. The current PMF simulation provides a quantitative description of the unfolding process of 15-TBA DNA driven by stretching and gives molecular insight on its detailed changes of base pair interactions in the presence of the metal cation.

Free-Energy Landscape of a pH-Modulated G·C Base Pair Transition from Watson-Crick to Hoogsteen State in Duplex DNA.

In double-helical DNAs, the most stable Watson-Crick (WC) base pair (bp) can be in thermal equilibrium with much less abundant Hoogsteen (HG) bp by the spontaneous rotation of the glycosidic angle in purine bases. Previous experimental studies showed that in the case of a G·C bp, the population of the transient HG is enhanced as a protonated form (HG+) through the protonation of the cytosine base under weakly acidic conditions. Hence, pH is a key factor that can modulate this transition event from the WC to HG+ bp. In this study, to computationally probe the overall free-energy landscapes of this pH-modulated G·C HG breathing, a comprehensive classical molecular dynamics (MD) simulation protocol is proposed using an enhanced sampling MD in conjunction with the standard thermodynamic integration method. From this MD protocol proposed, the free-energy surfaces of the G·C bp transition from the WC to HG bp were constructed successfully at any pH range, producing pH-dependent free-energy quantities in close agreement with previously reported experimental results. The simulation protocol is expected to provide valuable atomistic insight into the DNA bp transition events coupled with protonation or tautomeric shift in a target bp.

Computational Probing of Temperature-Dependent Unfolding of a Small Globular Protein: From Cold to Heat Denaturation.

Fully atomistic replica exchange molecular dynamics simulations are performed to compute the stability curve of a small globular protein as accurately as possible. To investigate the individual roles of the protein and water parts, we compute the conformational entropy change of this protein directly from the simulation ensembles. This entropy calculation enables complete separations of the unfolding changes of enthalpy and the entropy into their own protein and hydration components. From this decomposition, we are able to determine the main thermodynamic factors governing the cold and heat unfolding events: the cold and heat unfolding events are largely driven by the hydration enthalpy gain and the protein conformational entropy gain, respectively. This computational study discloses several temperature-dependent unfolding thermodynamic behaviors of the protein and water compartments and establishes their unique relationship. Upon unfolding, the changes of enthalpy and entropy in the protein part are all positive convex functions of temperature, whereas the equivalent changes in the water part are all negative concave functions of temperature. Hence, these two mutually opposing effects from the protein and water parts dictate the thermodynamics of unfolding. Furthermore, consistent with the temperature-dependent behaviors of the protein part, the changes of the solvent-accessible surface area and the radius of gyration of the protein upon unfolding are also convex functions of temperature. Hence, all these new temperature dependences, combined together, pave the way to unveiling the thermodynamic and structural features of protein denaturation events at various temperature conditions.

Improving Temperature Generator in Parallel Tempering Simulation in the NPT Condition.

Parallel tempering (PT) simulations provide a powerful computational tool to accelerate conformational searches of complex molecular systems. In this method, multiple replica systems assigned to different temperatures run in parallel and undergo tandem replica exchange events to enhance conformational mixing in temperature space. Efficient PT simulations require a uniform acceptance ratio across all the replicas. In the context of a previous energy distribution-based temperature generation (TG) scheme, we propose an improved TG protocol to maintain such a uniform exchange probability in general PT simulations.

Conformer-Specific Photodissociation Dynamics of CF2ICF2I in Solution Probed by Time-Resolved Infrared Spectroscopy.

The photodissociation dynamics of CF2ICF2I in solution was investigated from 0.3 ps to 100 µs, after the excitation of CF2ICF2I with a femtosecond UV pulse. Upon excitation, one I atom is eliminated within 0.3 ps, producing a haloethyl radical having a classical structure: anti-CF2ICF2 and gauche-CF2ICF2. All the nascent gauche-CF2ICF2 radicals reacted with the dissociated I atom within the solvent cage to produce a complex, I2··C2F4, in <1 ps. The quasi-stable I2··C2F4 complex in CCl4 (CH3CN or CD3OH) further dissociated into I2 and C2F4 with a time constant of 180 ± 5 (46 ± 3) ps. Some of the anti-CF2ICF2 radicals also formed the I2··C2F4 complex with a time constant of 1.5 ± 0.3 ps, while the remaining radicals underwent secondary elimination of I atom in a few nanoseconds. The time constant for the secondary dissociation of I atom from the anti-CF2ICF2 radical was independent of the excitation wavelength, indicating that the excess energy in the nascent radical is relaxed and that the secondary dissociation proceeds thermally. The formation of the I2··C2F4 complex and the thermal dissociation of the anti-CF2ICF2 radical clearly demonstrate that even a weakly interacting solvent plays a significant role in the modification and creation of reaction.

All-atom ab initio native structure prediction of a mixed fold (1FME): a comparison of structural and folding characteristics of various beta beta alpha miniproteins.

We performed an all-atom ab initio native structure prediction of 1FME, which is one of the computationally challenging mixed fold beta beta alpha miniproteins, by combining a novel conformational search algorithm (multiplexed Q-replica exchange molecular dynamics scheme) with a well-balanced all-atom force field employing a generalized Born implicit solvation model (param99MOD5/GBSA). The nativelike structure of 1FME was identified from the lowest free energy minimum state and in excellent agreement with the NMR structure. Based on the interpretation of the free energy landscape, the structural properties as well as the folding behaviors of 1FME were compared with other beta beta alpha miniproteins (1FSD, 1PSV, and BBA5) that we have previously studied with the same force field. Our simulation showed that the 28-residue beta beta alpha miniproteins (1FME, 1FSD, and 1PSV) share a common feature of the free energy topography and exhibit the three local minimum states on each computed free energy map, but the 23-residue miniprotein (BBA5) follows a downhill folding with a single minimum state. Also, the structure and stability changes resulting from the two point mutation (Gln1-->Glu1 and Ile7-->Tyr7) of 1FSD were investigated in details for direct comparison with the experiment. The comparison shows that upon mutation, the experimentally observed turn type switch from an irregular turn (1FSD) to type I(') turn (1FME) was well reproduced with the present simulation.

Computational Probing of Watson-Crick/Hoogsteen Breathing in a DNA Duplex Containing N1-Methylated Adenine.

DNA breathing is a local conformational fluctuation spontaneously occurring in double-stranded DNAs. In particular, the possibility of individual base pairs (bps) in duplex DNA to flip between alternate bp modes, i.e., Watson-Crick (WC)-like and Hoogsteen (HG)-like, at relevant time scales has impacted DNA research fields for many years. In this study, to computationally probe effects of chemical modification on the DNA breathing, we present a free energy landscape of spontaneous thermal transitions between WC and HG bps in a free DNA duplex containing N1-methylated adenine (m1A). For the current free energy computation, a variant of well-tempered metadynamics simulation was extensively performed for a total of 40 µs to produce free energy surfaces. The free energy profile indicated that, upon the chemical modification of adenine, the HG bp (m1A·T) was located in the most favorable conformation (96.7%); however, the canonical WC bp (m1A·T) was distorted into two WC-like bps of WC* (2.8%) and WC** (0.5%). The conformational exchange between these two minor WC-like bps occurs with the first hundred nanoseconds. The transition between WC-like and HG bp features multiple transition pathways displaying various extents of base flipping in combination with glycosidic rotation. Analysis of the simulated ensemble showed that the m1A-induced changes of the backbone and sugar pucker were in a reasonable agreement with previous results inferred from NMR experiments. Also, this study revealed that the formation of the stable HG bp upon the mutation alters the characteristics of dynamic fluctuations of the neighboring WC residues of m1A. We expect this simulation approach to be a robust computational scheme to complement and guide future high-resolution experiments on many outstanding issues of duplex DNA breathing.

Free energy landscapes of a highly structured beta-hairpin peptide and its single mutant.

We investigated the free energy landscapes of a highly structured beta-hairpin peptide (MBH12) and a less structured peptide with a single mutation of Tyr6 to Asp6 (MBH10). For the free energy mapping, starting from an extended conformation, the replica exchange molecular dynamic simulations for two beta-hairpins were performed using a modified version of an all-atom force field employing an implicit solvation (param99MOD5/GBSA). With the present simulation approach, we demonstrated that detailed stability changes associated with the sequence modification from MBH12 to MBH10 are quantitatively well predicted at the all-atom level.

Direct folding simulation of alpha-helices and beta-hairpins based on a single all-atom force field with an implicit solvation model.

Recently, we have shown that a modified energy model based on the param99 force field with the generalized Born (GB) solvation model produces reliable free energy landscapes of mini-proteins with a betabetaalpha motif (BBA5, 1FSD, and 1PSV), with the native structures of the mini-proteins located in their lowest free energy minimum states. One of the main features in the modified energy model is a significant improvement for more balanced treatments of alpha and beta strands in proteins. In this study, using the replica exchange molecular dynamics (REMD) simulation method with this new force field, we have carried out extensive ab initio folding studies of several well-known peptides with alpha or beta strands (C-peptide, EK-peptide, le0q, and gbl). Starting from fully extended conformations as the initial conditions, all of the native-like structures of the target peptides were successfully identified by REMD, with reasonable representations of free energy surfaces. The present simulation results with the modified energy model are consistent with experiments, demonstrating an extended applicability of the energy model to folding studies of a variety of alpha-helices, beta-strands, and alpha/beta proteins.

Predicting RNA Structures via a Simple van der Waals Correction to an All-Atom Force Field.

We proposed a simple van der Waals backbone correction (O2' and OP) to the AMBER ff12 force field in conjunction with the OPC water via an unequal Lorentz-Berthelot combination rule. As tested on four different tetranuceotides such as r(GACC), r(CCCC), r(AAAA), and r(CAAU), this new force field correctly captured each native fold as the largest population. For a RNA tetraloop (UUCG) tested, the stability of its native fold is substantially improved.