Conformational Relaxation Research Articles

Simplified qPCR method for detecting excessive mtDNA damage induced by exogenous factors.

Damage to mitochondrial DNA (mtDNA) is a meaningful biomarker for evaluating genotoxicity of drugs and environmental toxins. Existing PCR methods utilize long mtDNA fragments (∼8-10kb), which complicates detecting exact sites of mtDNA damage. To identify the mtDNA regions most susceptible to damage, we have developed and validated a set of primers to amplify ∼2kb long fragments, while covering over 95% of mouse mtDNA. We have modified the detection method by greatly increasing the enrichment of mtDNA, which allows us solving the problem of non-specific primer annealing to nuclear DNA. To validate our approach, we have determined the most damage-susceptible mtDNA regions in mice treated in vivo and in vitro with rotenone and H2O2. The GTGR-sequence-enriched mtDNA segments located in the D-loop region were found to be especially susceptible to damage. Further, we demonstrate that H2O2-induced mtDNA damage facilitates the relaxation of mtDNA supercoiled conformation, making the sequences with minimal damage more accessible to DNA polymerase, which, in turn, results in a decrease in threshold cycle value. Overall, our modified PCR method is simpler and more selective to the specific sites of damage in mtDNA.

Single-Molecule Investigations of Conformation Adaptation of Porphyrins on Surfaces.

The porphyrin macrocyclic core features dynamic conformational transformations in free space because of its structural flexibility. Once attached to a substrate, the molecule-substrate interaction often restricts this flexibility and stabilizes the porphyrin in a specific conformation. Here using molecular dynamic and density-functional theory simulations and scanning tunneling microscopy and spectroscopy, we investigated the conformation relaxation and stabilization processes of two porphyrin derivatives (5,15-dibromophenyl-10,20-diphenylporphyrin, Br2TPP, and 5,15-diphenylporphyrin, DPP) adsorbed on Au(111) and Pb(111) surfaces. We found that Br2TPP adopts either dome or saddle conformations on Au(111) but only the saddle conformation on Pb(111), whereas DPP deforms to a ruffled conformation on Au(111). We also resolved the structural transformation pathway of Br2TPP from the free-space conformations to the surface-anchored conformations. These findings provide unprecedented insights revealing the conformation adaptation process. We anticipate that our results may be useful for controlling the conformation of surface-anchored porphyrin molecules.

Depth-resolved local conformation and thermal relaxation of polystyrene near substrate interface.

By means of sum-frequency generation spectroscopy, we report a depth-resolved measurement of the local conformation and chain relaxation of polystyrene (hPS) located at different distances from the quartz interface. To control the distance from the quartz interface, deuterated polystyrene (dPS) layers with thicknesses of 3.4, 7.5, and 20 nm were coated on the quartz substrates. The hPS chains in direct contact with the substrate surface predominantly orient their phenyl rings in a direction normal to the substrate. This conformation was found to be barely relaxed when the film was annealed for 24 h at 423 K, higher than the bulk glass transition temperature. In contrast, for the hPS chains supported on the dPS layer, the orientation of phenyl rings of hPS became weaker with the annealing and this trend was more significant with increasing distance from the quartz substrate. In particular, the orientation of phenyl rings of hPS after annealing vanished at a distance of 20 nm. These results might provide an important evidence of the difference in the relaxation dynamics of the PS chains located at different distances from the quartz interface.

Ultrafast investigation of photoinduced charge transfer in aminoanthraquinone pharmaceutical product

We investigated the mechanism of intramolecular charge transfer and the following radiationless dynamics of the excited states of 1-aminoanthraquinone using steady state and time-resolved absorption spectroscopy combined with quantum chemical calculations. Following photoexcitation with 460 nm, conformational relaxation via twisting of the amino group, charge transfer and the intersystem crossing (ISC) processes have been established to be the major relaxation pathways responsible for the ultrafast nonradiative of the excited S1 state. Intramolecular proton transfer, which could be induced by intramolecular hydrogen bonding is inspected and excluded. Time-dependent density functional theory (TDDFT) calculations reveal the change of the dipole moments of the S0 and S1 states along the twisted coordinate of the amino group, indicating the mechanism of twisted intra-molecular charge transfer (TICT). The timescale of TICT is measured to be 5 ps due to the conformational relaxation and a barrier on the S1 potential surface. The ISC from the S1 state to the triplet manifold is a main deactivation pathway with the decay time of 28 ps. Our results observed here have yield a physically intuitive and complete picture of the photoinduced charge transfer and radiationless dynamics in anthraquinone pharmaceutial products.

Computer simulation studies of the influence of side alkyl chain on glass transition behavior of carbazole trimer

In this work, all-atom molecular dynamics simulations were employed to study the influence of the side alkyl chain on glass transition behavior of several carbazole trimers (CT) in a temperature range from 423 to 183 K. The glass transition temperatures were obtained from the break in the slope of the volume-temperature curves and found to agree with the experimental values. The short time dynamics of four CT molecules were probed by using velocity autocorrelation functions and mean-square displacements. The current studies showed that the dynamics of CT systems can be easily interpreted through the cage effect. Furthermore, the investigation of the torsional autocorrelation function and P2-state/P3-state functions showed that the rotational barriers of side chains can slow down the conformational relaxation and lead to stronger temperature dependence of conformational relaxation. The relaxation time, characteristic time of P2-state(t) and P3-state(t) functions were all found to have Arrhenius-type temperature dependence.

Reaction Path Averaging: Characterizing the Structural Response of the DNA Double Helix to Electron Transfer.

A polarizable environment, prominently the solvent, responds to electronic changes in biomolecules rapidly. The knowledge of conformational relaxation of the biomolecule itself, however, may be scarce or missing. In this work, we describe in detail the structural changes in DNA undergoing electron transfer between two adjacent nucleobases. We employ an approach based on averaging of tens to hundreds of thousands of nonequilibrium trajectories generated with molecular dynamics simulation, and a reduction of dimensionality suitable for DNA. We show that the conformational response of the DNA proceeds along a single collective coordinate that represents the relative orientation of two consecutive base pairs, namely, a combination of helical parameters shift and tilt. The structure of DNA relaxes on time scales reaching nanoseconds, contributing marginally to the relaxation of energies, which is dominated by the modes of motion of the aqueous solvent. The concept of reaction path averaging (RPA), conveniently exploited in this context, makes it possible to filter out any undesirable noise from the nonequilibrium data, and is applicable to any chemical process in general.

Fibrillation-prone conformations of the amyloid-β-42 peptide at the gold/water interface

Proteins in the proximity of inorganic surfaces and nanoparticles may undergo profound adjustments that trigger biomedically relevant processes, such as protein fibrillation. The mechanisms that govern protein-surface interactions at the molecular level are still poorly understood. In this work, we investigate the adsorption onto a gold surface, in water, of an amyloid-β (Aβ) peptide, which is the amyloidogenic peptide involved in Alzheimer's disease. The entire adsorption process, from the peptide in bulk water to its conformational relaxation on the surface, is explored by large-scale atomistic molecular dynamics (MD) simulations. We start by providing a description of the conformational ensemble of Aβ in solution by a 22 μs temperature replica exchange MD simulation, which is consistent with previous results. Then, we obtain a statistical description of how the peptide approaches the gold surface by multiple MD simulations, identifying the preferential gold-binding sites and giving a kinetic picture of the association process. Finally, relaxation of the Aβ conformations at the gold/water interface is performed by a 19 μs Hamiltonian-temperature replica exchange MD simulation. We find that the conformational ensemble of Aβ is strongly perturbed by the presence of the surface. In particular, at the gold/water interface the population of the conformers akin to amyloid fibrils is significantly enriched, suggesting that this extended contact geometry may promote fibrillation.

Microscopic nucleation and propagation rates of an alanine-based α-helix.

An infrared temperature-jump (T-jump) study by Huang et al. (Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 2788-2793) showed that the conformational relaxation kinetics of an alanine-based α-helical peptide depend not only on the final temperature (Tf) but also on the initial temperature (Ti) when Tf is fixed. Their finding indicates that the folding free energy landscape of this peptide is non-two-state like, allowing for the population of conformational ensembles with different helical lengths and relaxation times in the temperature range of the experiment. Because α-helix folding involves two fundamental events, nucleation and propagation, the results of Huang et al. thus present a unique opportunity to determine their rate constants - a long-sought goal in the study of the helix-coil transition dynamics. Herein, we capitalize on this notion and develop a coarse-grained kinetic model to globally fit the thermal unfolding curve and T-jump kinetic traces of this peptide. Using this strategy, we are able to explicitly determine the microscopic rate constants of the kinetic steps encountered in the nucleation and propagation processes. Our results reveal that the time taken to form one α-helical turn (i.e., an α-helical segment with one helical hydrogen bond) is about 315 ns, whereas the time taken to elongate this nucleus by one residue (or backbone unit) is 5.9 ns, depending on the position of the residue.

Luminescence Switching of a Gold–Copper Supramolecular Complex: A Physical Insight

Stimuli-responsive metal complexes are extensively studied due to their promising properties for various practical applications. In many cases, stimuli-induced response is provoked by phase transitions, which completely modify electronic properties of chromophoric center to give substantial modification of the phase emissive properties. However, complexity of the systems hinders theoretical calculations, which could be used to interpret the emission response, even at qualitative level. In this study, we apply a joint experimental and theoretical approach to model the luminescence switching of a vapochromic Au–Cu organometallic supramolecular complex in solution, amorphous, and crystal state. We have investigated luminescence spectra and emission anisotropy of the complex in solution, polymer gel, amorphous, and crystal states. The excitation and emission energies of the complex in vacuum and crystal state have been calculated using QM and QM/MM approaches. The results show that (1) the red shift of emission of the complex in solution is caused by the conformational relaxation in the T1 state and (2) the blue shift of crystalline state emission originates from the interactions of the complex with the crystal environment. The model elaborated to describe the phenomena observed may provide a practical way to analyze stimuli-induced luminescence switching in analogous systems.

Photophysical Study of Twop-Phenylenevinylene Oligomers End-Capped with Triphenylamine or Cyanoacetate Groups.

The photophysical properties of two p-phenylenevinylene trimer end-capped with triphenylamine or cyanoacetate groups were investigated with special attention to the different donor-acceptor character of the terminal units, relatively to a same central phenylenevinylene trimer. It is shown that these conjugated small molecules constitute a quite useful model system to understand how the end-capped groups couple to the central molecular unit with respect to energy and/or electron transfer. The study of these two compounds in solution showed that after photoexcitation, the conformational relaxation leads to an increase in the conjugated backbone planarization. A more pronounced intramolecular charge transfer in the excited state of the molecule with two alkyl cyanoacetate groups as electron-withdrawing ends was also found. The molecular structures of the two compounds are especially advantageous for electronic applications such as organic photovoltaics, for example, due to their narrow optical band gaps that potentially lead to a more efficient photon harvesting in the organic devices fabricated with these compounds.

Exploring Electron/Proton Transfer and Conformational Changes in the Nitrogenase MoFe Protein and FeMo-cofactor Through Cryoreduction/EPR Measurements.

We combine cryoreduction/annealing/EPR measurements of nitrogenase MoFe protein with results of earlier investigations to provide a detailed view of the electron/proton transfer events and conformational changes that occur during early stages of [e-/H+] accumulation by the MoFe protein. This includes reduction of (i) the non-catalytic state of the iron-molybdenum cofactor (FeMo-co) active site that is generated by chemical oxidation of the resting-state cofactor (S = 3/2)) within resting MoFe (E0), and (ii) the catalytic state that has accumulated n =1 [e-/H+] above the resting-state level, denoted E1(1H) (S ≥ 1) in the Lowe-Thorneley kinetic scheme. FeMo-co does not undergo a major change of conformation during reduction of oxidized FeMo-co. In contrast, FeMo-co undergoes substantial conformational changes during the reduction of E0 to E1(1H), and of E1(1H) to E2(2H) (n = 2; S = 3/2). The experimental results further suggest that the E1(1H) → E2(2H) step involves coupled delivery of a proton and electron (PCET) to FeMo-co of E1(H) to generate a non-equilibrium S = ½ form E2(2H)*. This subsequently undergoes conformational relaxation and attendant change in FeMo-co spin state, to generate the equilibrium E2(2H) (S = 3/2) state. Unexpectedly, these experiments also reveal conformational coupling between FeMo-co and P-cluster, and between Fe protein binding and FeMo-co, which might play a role in gated ET from reduced Fe protein to FeMo-co.

Excited-State Proton Transfer and Conformational Relaxation of 2-(4'-Pyridyl)benzimidazole in Nafion Films.

The effect of annealing on the acidity and water uptake of Nafion films has been studied by using the acidity sensing fluorophore 2-(4'-pyridyl)benzimidazole (4PBI). The difference in acidity and the microenvironment of the fluorophore in annealed and nonannealed films is brought out in this study. The annealed film is found to have less water uptake than nonannealed films. The amount of water uptake increases upon acid treatment of the films, as all the steady-state and time-resolved behaviour of the molecule in nonannealed films is restored. These observations are rationalised by the formation of anhydrides upon annealing and their hydrolysis to sulfonic acid groups upon acid treatment. Interestingly, the acidity of annealed films is found to be even less than that of Na+ exchanged films, indicating that annealing removes more protons from the Nafion films than cation exchange can.

Relaxation folding and the principle of the minimum rate of energy dissipation for conformational motions in a viscous medium

A numerical simulation of the folding of a model polymer chain of 50 units with valence bonds of a fixed length and fixed valence angle values has been performed using the strong friction approximation. The rate of energy dissipation in the system has been analyzed for conformational motions along a trajectory determined by the equations of mechanics and the trajectories characterized by random and variable deviations from the mechanical path. The validity of the principle of the minimum average rate of the energy dissipation for the conformational relaxation of a macromolecule in a viscous medium has been demonstrated. A profile of the relaxation energy funnel for the folding of a macromolecular chain has been constructed. Slow and rapid stages of folding could be distinguished in the energy funnel profile; the final state was separated from the nearest conformations of the folded chain by an energy gap.

Direct Time‐Domain Observation of Conformational Relaxation in Gas‐Phase Cold Collisions

AbstractCooling molecules in the gas phase is important for precision spectroscopy, cold molecule physics, and physical chemistry. Measurements of conformational relaxation cross sections shed important light on potential energy surfaces and energy flow within a molecule. However, gas‐phase conformational cooling has not been previously observed directly. In this work, we directly observe conformational dynamics of 1,2‐propanediol in cold (6 K) collisions with atomic helium using microwave spectroscopy and buffer‐gas cooling. Precise knowledge and control of the collisional environment in the buffer‐gas allows us to measure the absolute collision cross‐section for conformational relaxation. Several conformers of 1,2‐propanediol are investigated and found to have relaxation cross‐sections with He ranging from σ=4.7(3.0)×10−18 cm2 to σ>5×10−16 cm2. Our method is applicable to a broad class of molecules and could be used to provide information about the potential energy surfaces of previously uninvestigated molecules.

Direct Time-Domain Observation of Conformational Relaxation in Gas-Phase Cold Collisions.

Cooling molecules in the gas phase is important for precision spectroscopy, cold molecule physics, and physical chemistry. Measurements of conformational relaxation cross sections shed important light on potential energy surfaces and energy flow within a molecule. However, gas-phase conformational cooling has not been previously observed directly. In this work, we directly observe conformational dynamics of 1,2-propanediol in cold (6 K) collisions with atomic helium using microwave spectroscopy and buffer-gas cooling. Precise knowledge and control of the collisional environment in the buffer-gas allows us to measure the absolute collision cross-section for conformational relaxation. Several conformers of 1,2-propanediol are investigated and found to have relaxation cross-sections with He ranging from σ=4.7(3.0)×10(-18) cm(2) to σ>5×10(-16) cm(2). Our method is applicable to a broad class of molecules and could be used to provide information about the potential energy surfaces of previously uninvestigated molecules.

USP7 Enforces Heterochromatinization of p53 Target Promoters by Protecting SUV39H1 from MDM2-Mediated Degradation.

The H3K9me3 repressive histone conformation of p53 target promoters is abrogated in response to p53 activation by MDM2-mediated SUV39H1 degradation. Here, we present evidence that the USP7 deubiquitinase protects SUV39H1 from MDM2-mediated ubiquitination in the absence of p53 stimulus. USP7 occupies p53 target promoters in unstressed conditions, a process that is abrogated with p53 activation associated with loss of the H3K9me3 mark on these same promoters. Mechanistically, USP7 forms a trimeric complex with MDM2 and SUV39H1, independent of DNA, and modulates MDM2-dependent SUV39H1 ubiquitination. Furthermore, we show that this protective function of USP7 on SUV39H1 is independent of p53. Finally, USP7 blocking cooperates with p53 in inducing apoptosis by enhancing p53 promoter occupancy and dependent transactivation of target genes. These results uncover a layer of the p53 transcriptional program mediated by USP7, which restrains relaxation of local chromatin conformation at p53 target promoters.

Dissipation in monotonic and non-monotonic relaxation to equilibrium.

Using molecular dynamics simulations, we study field free relaxation from a non-uniform initial density, monitored using both density distributions and the dissipation function. When this density gradient is applied to colour labelled particles, the density distribution decays to a sine curve of fundamental wavelength, which then decays conformally towards a uniform distribution. For conformal relaxation, the dissipation function is found to decay towards equilibrium monotonically, consistent with the predictions of the relaxation theorem. When the system is initiated with a more dramatic density gradient, applied to all particles, non-conformal relaxation is seen in both the dissipation function and the Fourier components of the density distribution. At times, the system appears to be moving away from a uniform density distribution. In both cases, the dissipation function satisfies the modified second law inequality, and the dissipation theorem is demonstrated.

Conformational plasticity of IgG during protein A affinity chromatography

Single step elution of a protein A column with 100mM acetate pH 3.5 produced a curvilinear gradient with pH dropping steeply at first then more gradually as it approached endpoint. IgG with a native hydrodynamic diameter of 11.5nm began to elute at pH 6.0 with a size of 9.4nm. IgG size continued to decrease across the peak, reaching a minimum of 2.2nm at pH 3.9. Secondary structure of early eluting IgG was only mildly affected but later eluting fractions became increasingly non-native with the 2.2nm population exhibiting the highest proportion of β-sheet and lowest random coil of all conformations. Size reduction and structural change of IgG through this portion of the elution peak were attributed dominantly to a pre-existing tendency of highly concentrated IgG to adopt reduced size conformations at low pH and conductivity, facilitated by the known conformational relaxation of IgG by its interaction with protein A. IgG size increased to 10.4nm as elution pH approached 3.5 across the tailing fractions. Major loss of β-sheet and increase of α-helix and random coil were observed in parallel. Late elution of this population was attributed to it being eluted from interactions with 2 distinct protein A domains, one bound to each side of the Fc region, creating a higher dissociation constant than single-site Fc-protein A interactions, and requiring more severely disruptive conditions for elution. The high degree of conformational disruption was attributed to simultaneous interaction of both heavy chains with protein A.

Excited-state intramolecular proton transfer and conformational relaxation in 4'-N,N-dimethylamino-3-hydroxyflavone doped in acetonitrile crystals.

The effect of intermolecular interactions on excited-state intramolecular proton transfer (ESIPT) in 4'-N,N-dimethylamino-3-hydroxyflavone (DMHF) doped in acetonitrile crystals was investigated by measuring its temperature dependence of steady-state fluorescence excitation and fluorescence spectra and picosecond time-resolved spectra. The relative intensity of emission from the excited state of the normal form (N*) to that from the excited state of the tautomer form (T*) and spectral features changed markedly with temperature. Unusual changes in the spectral shift and spectral features were observed in the fluorescence spectra measured between 200 and 218 K, indicating that a solid-solid phase transition of DMHF-doped acetonitrile crystals occurred. Time-resolved fluorescence spectra suggested conformational relaxation of the N* state competed with ESIPT after photoexcitation and the ESIPT rate increased with temperature in the low-temperature phase of acetonitrile. However, the intermolecular interaction of N* with acetonitrile in the high-temperature phase markedly stabilized the potential minimum of the fluorescent N* state and slowed the ESIPT. This stabilization can be explained by reorganization of acetonitrile originating from the strong electric dipole-dipole interaction between DMHF and acetonitrile molecules.

Continuum Electrostatics Approaches to Calculating pKas and Ems in Proteins.

Proteins change their charge state through protonation and redox reactions as well as through binding charged ligands. The free energy of these reactions is dominated by solvation and electrostatic energies and modulated by protein conformational relaxation in response to the ionization state changes. Although computational methods for calculating these interactions can provide very powerful tools for predicting protein charge states, they include several critical approximations of which users should be aware. This chapter discusses the strengths, weaknesses, and approximations of popular computational methods for predicting charge states and understanding the underlying electrostatic interactions. The goal of this chapter is to inform users about applications and potential caveats of these methods as well as outline directions for future theoretical and computational research.