Structural impacts of two disease-linked ADAR1 mutants: a molecular dynamics study.

Adenosine deaminases acting on RNA (ADARs) are pivotal RNA-editing enzymes responsible for converting adenosine to inosine within double-stranded RNA (dsRNA). Dysregulation of ADAR1 editing activity, often arising from genetic mutations, has been linked to elevated interferon levels and the onset of autoinflammatory diseases. However, understanding the molecular underpinnings of this dysregulation is impeded by the lack of an experimentally determined structure for the ADAR1 deaminase domain. In this computational study, we utilized homology modeling and the AlphaFold2 to construct structural models of the ADAR1 deaminase domain in wild-type and two pathogenic variants, R892H and Y1112F, to decipher the structural impact on the reduced deaminase activity. Our findings illuminate the critical role of structural complementarity between the ADAR1 deaminase domain and dsRNA in enzyme-substrate recognition. That is, the relative position of E1008 and K1120 must be maintained so that they can insert into the minor and major grooves of the substrate dsRNA, respectively, facilitating the flipping-out of adenosine to be accommodated within a cavity surrounding E912. Both amino acid replacements studied, R892H at the orthosteric site and Y1112F at the allosteric site, alter K1120 position and ultimately hinder substrate RNA binding.

Studies on lysozyme modifications induced by substituted p-benzoquinones.

Protein misfolding can facilitate a protein damaging process and makes it susceptible to a series of events such as unfolding, adduct formation, oligomerization, or aggregation. Loss of a protein's native structure may result in its biological malfunction and/or cellular toxicity that could cause associated diseases. Several factors were identified for causing structural changes of a protein, however quinone-induced protein modifications received very little attention whether for amyloidal or non-amyloidal proteins. In this paper, we report our investigation on lysozyme modifications upon treatment with selected benzoquinones (BQs), utilizing fluorescence spectroscopy including anisotropy determination, UV-Vis spectroscopy, and SDS-PAGE. Lysozyme was reacted with substituted BQs in order to examine substituent effects on protein modifications. In addition, we evaluated lysozyme modifications induced by 1,4-benzoquinone in concentration-, pH-, temperature-, and time-dependent studies. Our study shows that all BQs can readily modify lysozyme in a complex manner through adduct formation, oligomerization, polymeric aggregation, and/or fibrilization. Electrochemical properties of selected BQs were monitored using cyclic voltammetry in phosphate buffered aqueous solution, and it was found that quinone reduction potentials correlate well with their reactivity trend toward lysozyme.

A comparison study on ribonuclease A modifications induced by substituted p-benzoquinones.

In this paper, we present our investigation on ribonuclease A (RNase) modifications induced by 1,4-benzoquinone (PBQ), 2-methyl-1,4-benzoquinone (MBQ), and 2-chloro-1,4-benzoquinone (CBQ). The goal of the study was to evaluate quinone-induced protein modifications as well as substituent effects, utilizing several techniques such as SDS-PAGE, fluorescence spectroscopy, microscopy, and LC-ESI(+)-QTOF-MS. SDS-PAGE experiments revealed that all quinones modify RNase through oligomerization as well as polymeric aggregation; with CBQ functioning as the most efficient quinone while MBQ was least efficient. The fluorescence emission was found to be less intense and the anisotropy values were found to be slightly higher for the modified RNase compared to the unmodified RNase. UV-Vis spectroscopy indicated that all three quinones formed adducts in which they were covalently linked to RNase. Confocal imaging analysis showed that the presence of CBQ resulted in massive RNase aggregation, while PBQ-treated RNase formed much smaller aggregates. MBQ-treated RNase exhibited micrographic features that closely resembled those of the unmodified RNase. LC-ESI(+)-QTOF-MS studies indicated the nature of PBQ- and CBQ-induced RNase modifications are complex mainly due to simultaneously occurrence of both adduct formation and oligomerization. Kinetic studies on quinone reactivity toward lysine revealed the rank order of CBQ>PBQ≫MBQ, based on the second-order rate constants. We also utilized scanning electron microscopy in order to investigate the effect of modified RNase on the biomineralization of salts.

Modifications of ribonuclease A induced by p-benzoquinone.

The nature of ribonuclease A (RNase) modifications induced by p-benzoquinone (pBQ) was investigated using several analysis methods. SDS-PAGE experiments revealed that pBQ was efficient in producing oligomers and polymeric aggregates when RNase was incubated with pBQ. The fluorescence behavior and anisotropy changes of the modified RNase were monitored for a series of incubation reactions where RNase (0.050 mM) was incubated with pBQ (0.050, 0.25, 0.50, 1.50 mM) at 37 °C in phosphate buffer (pH 7.0, 50 mM). The modified RNase exhibited less intense fluorescence and slightly higher anisotropy than the unmodified RNase. UV-Vis spectroscopy indicated that pBQ formed covalent bonds to the modified RNase. Confocal imaging analysis confirmed the formation of the polymeric RNase aggregates with different sizes upon exposure of RNase to high concentrations of pBQ. The interaction between the modified RNase and salts affecting biomineralization of salts was also investigated by scanning electron microscopy. Overall, our results show that pBQ can induce formation of both RNase adducts and aggregates thus providing a better understanding of its biological activity.

Hybrid density functional theory with a specific reaction parameter: hydrogen abstraction reaction of difluoromethane by the hydroxyl radical.

Accurate potential energy surfaces for the OH + CH2F2 --> H2O + CHF2 reaction are constructed using hybrid and hybrid meta density functional theory methods (mPW1PW91, B1B95, and mPW1B95) with specific reaction parameters in conjunction with the 6-31 + G(d,p) basis set. The accuracy of a surface is examined by comparing the calculated rate constants with the experimental ones. The rate constants are calculated over the temperature range 200-1,500 K using variational transition state theory with multidimensional tunneling contributions. The hybrid density functional theory methods with specific-reaction-parameter Hartree-Fock exchange contributions (39.2-41.0% for mPW1PW91, 41.0-42.2% for B1B95, and 44.9-46.3% for mPW1B95, respectively) provide accurate rate constants over an extended temperature range. The classical barrier height for the hydrogen abstraction reaction on these potential energy surfaces is determined to be 5.0-5.3 kcal mol(-1), and the best estimate value is 5.14 kcal mol(-1).

Association patterns in (HF)(m)(H2O)(n) (m + n = 2-8) clusters.

In an attempt to understand the phase behavior of aqueous hydrogen fluoride, the clustering in the mixture is investigated at the molecular level. The study is performed at the mPW1B95/6-31+G(d,p) level of theory. Several previous studies attempted to describe the dissociation of HF in water, but in this investigation, the focus is only on the association patterns that are present in this binary mixture. A total of 214 optimized geometries of (HF)n(H2O)m clusters, with m + n as high as 8, were investigated. For each cluster combination, several different conformations are investigated, and the preferred conformations are presented. Using multiple linear regressions, the average strengths of the four possible H-bonding interactions are obtained. The strongest H-bond interaction is reported to be the H2O...H-F interaction. The most probable distributions of mixed clusters as a function of composition are also deduced. It is found that the larger (HF)n(H2O)m clusters are favored both energetically and entropically compared to the ones that are of size m + n < or = 3. Also, the clusters with equimolar contributions of HF and H2O are found to have the strongest interactions.

Hybrid density functional theory with specific reaction parameter: hydrogen abstraction reaction of fluoromethane by the hydroxyl radical.

Three potential energy surfaces with specific reaction parameters are developed and tested for the OH + CH(3)F --> H(2)O + CH(2)F reaction. The goal of this work is to determine surfaces that provide calculated reaction rate constants that are comparable to the experimental data. The potential energy surfaces are constructed using hybrid and hybrid meta density functional theory methods, and the levels of electronic structure theory used in this study are mPW1PW91, B1B95, and mPW1B95 in conjunction with the 6-31+G(d,p) basis set. The reaction rate constants are calculated over the range 200-1,500 K using variational transition state theory with multidimensional tunneling contributions. New specific-reaction-parameter Hartree-Fock contributions are determined, and the hybrid density functional theory methods with these new contributions (35.5 +/- 1.2% for mPW1PW91, 36.6 +/- 1.2% for B1B95, and 40.7 +/- 1.2% for mPW1B95, respectively) reproduce accurate rate constants over an extended temperature range. On these potential energy surfaces, the classical barrier height for the hydrogen abstraction reaction is determined to be 3.4-3.8 kcal/mol, with a best estimate value of 3.6 kcal/mol.

Hybrid density functional theory study of fragment ions generated during mass spectrometry of 1,3-dioxane derivatives.

It was recently reported that the cis,cis and trans,trans diastereoisomers of four 2(r)-R-2,4(R),6(S)-trimethyl-1,3-dioxane derivatives show distinct electron ionization mass spectra. As a possible explanation for this finding, the authors suggested that the ions generated during the mass spectrometry of these compounds could follow different fragmentation patterns that initiate from different ion conformations. In this report, hybrid density functional theory methods have been used to investigate the conformational preference of three ions involved in the mass spectrometry of some 1,3-dioxane derivatives. We found that there is indeed more than one stable ion conformation for each of the investigated ions. Energy profiles along the torsional coordinates connecting the conformers are presented, and factors influencing the relative stability of ion conformations are discussed.

Carbon tunneling from a single quantum state.

We observed ring expansion of 1-methylcyclobutylfluorocarbene at 8 kelvin, a reaction that involves carbon tunneling. The measured rate constants were 4.0 x 10(-6) per second in nitrogen and 4 x 10(-5) per second in argon. Calculations indicated that at this temperature the reaction proceeds from a single quantum state of the reactant so that the computed rate constant has achieved a temperature-independent limit. According to calculations, the tunneling contribution to the rate is 152 orders of magnitude greater than the contribution from passage over the barrier. We discuss environmental effects of the solid-state inert-gas matrix on the reaction rate.