N1 Proton Research Articles

A pH‐Cascaded DNA Hydrogel Mediated by Reconfigurable A‐motif Duplex, i‐Motif Quadruplex, and T·A‐T Triplex Structures

AbstractGoverned by pH‐configurable A‐motif, i‐motif and T·A‐T triplex configurations, a cascaded DNA hydrogel subjecting to diverse pH values is presented. Under highly acidic conditions (pH 1.1), N1 protonation of adenine (pKa 3.5) in A‐strands generates AH+‐H+A units, resulting in a parallel A‐motif duplex crosslinked hydrogel. Under mild acid conditions (pH 5.2), the dissociation of A‐motif duplex into single A‐strands occurs due to the deprotonation of adenine, while N3 protonation of cytosine (pKa 6.5) in C‐strands creates hemi‐protonated C:C+ units, resulting in i‐motif bridged hydrogel. At neutral pH (pH 7.2), deprotonation of cytosine separates i‐motif crosslinkers. Simultaneously, the addition of auxiliary T‐strands results in the N3 protonation of thymine (pKa 10), generating T·A‐T triplex stabilized hydrogel. Under mildly alkaline conditions (pH 10.2), T·A‐T triplex separates into single T‐stands and A‐T duplex, resulting in the disassembly of DNA hydrogel. Therefore, a stepwise pH‐cascaded DNA hydrogel dictates the formation of structures following the pH steps 1.1 → 5.2 → 7.2 → 10.2 where pH‐triggered DNA secondary structures, including A‐motif, i‐motif and T·A‐T triplex, stabilize the hydrogel. The study advances DNA hydrogels from single pH‐responsiveness to multiple cascaded pH values, which opens up new possibilities for the development of smart hydrogels capable of adapting to various environmental conditions.

The photophysics of protonated cytidine and hemiprotonated cytidine base pair: A computational study.

We here study the effect that a lowering of the pH has on the excited state processes of cytidine and a cytidine/cytidine pair in solution, by integrating time-dependent density functional theory and CASSCF/CASPT2 calculations, and including solvent by a mixed discrete/continuum model. Our calculations reproduce the effect of protonation at N3 on the steady-state infrared and absorption spectra of a protonated cytidine (CH+ ), and predict that an easily accessible non-radiative deactivation route exists for the spectroscopic state, explaining its sub-ps lifetime. Indeed, an extremely small energy barrier separates the minimum of the lowest energy bright state from a crossing region with the ground electronic state, reached by out-of-plane motion of the hydrogen substituents of the CC double bond, the so-called ethylenic conical intersection typical of cytidine and other pyrimidine bases. This deactivation route is operative for the two bases forming an hemiprotonated cytidine base pair, [CH·C]+ , the building blocks of I-motif secondary structures, whereas interbase processes play a minor role. N3 protonation disfavors instead the nπ* transitions, associated with the long-living components of cytidine photoactivated dynamics.

Construction mechanisms of a new generation of high-energy all- and high-nitrogen materials under extreme conditions: Ab initio molecular dynamics simulations from pentazole compounds

State-of-the-art ab initio molecular dynamics (AIMD) simulations were performed to reveal the construction mechanisms of a new generation of high-energy all- and high-nitrogen materials under the extreme conditions of high temperature coupling of high pressure. The results indicate that there are two successive stages in the decomposition process for (N5-)2DABTT2+: (i) competition between distortion of cyclo-N5- and initial decomposition pathway of dehydrogenation of DABTT2+ cation; (ii) subsequent competition between ring-opening and protonation of N5-, as well as complex global decomposition reactions. Higher temperature or higher pressure accelerates the competition between distortion of cyclo-N5- and initial decomposition, and higher pressure makes distortion of cyclo-N5- dominant. N5- tends to protonate to form pentazole at 300 K, while there exists fierce competition between ring-opening of N5- and protonation of N5- at 1000 K, and higher pressure accelerates the competition and makes ring-opening of N5- dominant. Higher temperature or higher pressure leads to the earlier appearance of the largest nitrogen-rich cluster, and increasing either temperature or pressure could improve N content in the largest cluster. All- and high-nitrogen species N4, N6, HN5 and H2N6 were formed and crystal predictions were conducted. Our findings reveal the construction mechanisms of high-energy all-nitrogen materials with clean and pollution-free products under extreme conditions, which are of significance for the breakthrough of energy bottleneck in the preparation of all-nitrogen materials under conventional conditions, and provide an engineering solution for sustainable future of nature.

NEIL1 Recoding due to RNA Editing Impacts Lesion-Specific Recognition and Excision.

A-to-I RNA editing is widespread in human cells but is uncommon in the coding regions of proteins outside the nervous system. An unusual target for recoding by the adenosine deaminase ADAR1 is the pre-mRNA of the base excision DNA repair enzyme NEIL1 that results in the conversion of a lysine (K) to arginine (R) within the lesion recognition loop and alters substrate specificity. Differences in base removal by unedited (UE, K242) vs edited (Ed, R242) NEIL1 were evaluated using a series of oxidatively modified DNA bases to provide insight into the chemical and structural features of the lesion base that impact isoform-specific repair. We find that UE NEIL1 exhibits higher activity than Ed NEIL1 toward the removal of oxidized pyrimidines, such as thymine glycol, uracil glycol, 5-hydroxyuracil, and 5-hydroxymethyluracil. Gas-phase calculations indicate that the relative rates in excision track with the more stable lactim tautomer and the proton affinity of N3 of the base lesion. These trends support the contribution of tautomerization and N3 protonation in NEIL1 excision catalysis of these pyrimidine base lesions. Structurally similar but distinct substrate lesions, 5-hydroxycytosine and guanidinohydantoin, are more efficiently removed by the Ed NEIL1 isoform, consistent with the inherent differences in tautomerization, proton affinities, and lability. We also observed biphasic kinetic profiles and lack of complete base removal with specific combinations of the lesion and NEIL1 isoform, suggestive of multiple lesion binding modes. The complexity of NEIL1 isoform activity implies multiple roles for NEIL1 in safeguarding accurate repair and as an epigenetic regulator.

The N3/TiO2 interfacial structure is dependent on the pH conditions during sensitization.

The electronic structure of the N3/TiO2 interface can directly influence the performance of a dye sensitized solar cell (DSSC). Therefore, it is crucial to understand the parameters that control the dye's orientation on the semiconductor's surface. A typical step in DSSC fabrication is to submerge the nanoparticulate semiconductor film in a solution containing the dye, the sensitizing solution. The pH of the N3 sensitizing solution determines the distribution of the N3 protonation states that exist in solution. Altering the pH of the sensitizing solution changes the N3 protonation states that exist in solution and, subsequently, the N3 protonation states that anchor to the TiO2 substrate. We utilize the surface specific technique of heterodyne detected vibrational sum frequency generation spectroscopy to determine the binding geometry of N3 on a TiO2 surface as a function of the sensitizing solution pH conditions. It is determined that significant reorientation of the dye occurs in pH ≤2.0 conditions due to the lack of N3-dye carboxylate anchoring groups participating in adsorption to the TiO2 substrate. Consequently, the change in molecular geometry is met with a change in the interfacial electronic structure that can hinder electron transfer in DSSC architectures.

Steric and Electronic Interactions at Gln154 in ZEITLUPE Induce Reorganization of the LOV Domain Dimer Interface.

Plants measure light quality, intensity, and duration to coordinate growth and development with daily and seasonal changes in environmental conditions; however, the molecular details linking photochemistry to signal transduction remain incomplete. Two closely related light, oxygen, or voltage (LOV) domain-containing photoreceptor proteins, ZEITLUPE (ZTL) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), divergently regulate the protein stability of circadian clock and photoperiodic flowering components to mediate daily and seasonal development. Using structural approaches, we identified that mutations at the Gly46 position led to global rearrangements of the ZTL dimer interface in the isolated ZTL-LOV domain. Specifically, G46S and G46A variants induce a 180° rotation about the ZTL-LOV dimer interface that is coupled to ordering of N- and C-terminal signaling elements. These conformational changes hinge upon rotation of a C-terminal Gln residue (Gln154) analogous to that present in light-state structures of ZTL. In contrast to other LOV proteins, a Q154L variant retains light-state interactions with GIGANTEA (GI), thereby indicating N5 protonation is not required for ZTL signaling. The results presented herein confirm a divergent signaling mechanism within ZTL, whereby steric and electronic effects following adduct formation can be sufficient for signal propagation in LOV proteins containing a Gly residue at position 46. Examination of bacterial LOV structures with Gly residues at the equivalent position suggests that mechanisms of signal transduction in LOV proteins may be fluid across the LOV protein family.

Density Functional Theory-Based Molecular Modeling: Verification of Decisive Roles of Van der Waals Aggregation of Triiodide Ions for Effective Electron Transfer in Wet-Type N3-Dye-Sensitized Solar Cells

Density functional theory-based molecular modeling (DFT/MM) validates that KI and I2 undergo exothermic van der Waals (vdW) aggregation in acetonitrile (AN) or in the presence of 4-tert-butylpyridine (TBP), forming potassium triiodide (KI3) and, further mutual vdW aggregation leads to the formation of (KI3)2 and AN, (KI3)2 and (AN)2 and (KI3)2 and TBP in the AN-based Dye sensitized solar cells (DSSC) electrolytes. All KI3 aggregates have a very low energy gap, 0.17 eV, 0.14 eV and 0.05 eV of lowest unoccupied molecular orbital (LUMO) + 1 and LUMO, respectively, verifying efficient electron diffusion in μm-thick DSSC electrolytes. Hydrogen-bonding aggregation of anatase TiO2 model, Ti9O18H and OH, with N3 (proton) dye is also validated by DFT/MM, and the energy structure verifies unidirectional electron flow from highest occupied molecular orbital (HOMO) on thiocyanide (SCN) groups of N3 dye to LUMO on the TiO2 model at the aggregates. Further, DFT/MM for the aggregation of K+I3− with N3 verifies the most exothermic formation of the aggregate of N3 (proton) and K+I3−. The UV-Vis spectra of N3 (proton) and K+I3− is consistent with reported incident photocurrent efficiency (IPCE) action spectra (λ = 450–800 nm) of N3-sensitized DSSC, verifying that the N3 dye of N3 (proton) and K+I3− becomes an effective sensitizer in the anode / TiO2 / N3 (proton) / KI/I2 / acetonitrile (AN) / cathode structured DSSC. The energy structure of LUMO and LUMO + 1 of the aggregates, Ti9O18H and OH and N3 (proton), N3 and K+I3−, (KI3)2 and AN and (KI3)2 and TBP verifies high IPCE photocurrent and effective electron diffusion via KI3-aggregates in the DSSC of Ti9O18H and OH and N3 (proton) and K+I3−.

Electron and hole interactions with P, Z, and P:Z and the formation of mutagenic products by proton transfer reactions.

P and Z have recently been identified as promiscuous artificial nucleobases, which can behave as G and C, respectively, in duplex DNA. These nucleobases have been shown to participate in the replication reaction and can form stable B-DNA. A short sequence of DNA containing P and Z has also been shown to help in the diagnosis of diseases. However, the behavior of P and Z exposed to radiation has not been explored. As electrons and holes are created during the interaction of radiation with DNA bases, it is desirable to understand the electron or hole trapping abilities of P and Z in duplex DNA. To unravel these abilities, electron affinities (EAs) and ionization potentials (IPs) of P and Z in bare and microhydrated complexes are computed and compared with those of G and C by using the B3LYP-D3 dispersion-corrected density functional theory method and the IEFPCM method to account for the bulk solvation in water. The computed EA and IP values of P and Z are found to be largely positive and hence their anions (P˙- and Z˙-) and cations (P˙+ and Z˙+) would be stable in DNA. It is further found that the electron trapping ability of Z is significantly higher than that of P, G, and C. However, the hole trapping ability of P is slightly higher than that of Z, but less than that of G. To account for the proton transfer abilities of Z, Z˙+, and Z˙-, the stabilities of different proton transferred products and their tautomers are also explored. It is found that among the different products, the one formed by the transfer of the N3 proton would be the most stable. However, the N3 proton transfer from Z to P in the P:Z and P:Z˙- complexes would be unfeasible due to the high barrier and endothermic nature of the reaction. Remarkably, the same reaction in the P:Z˙+ complex is found to be exothermic with a low barrier energy. Hence, the conversion of Z to Z˙+ would facilitate N3 proton transfer from Z to P in the P:Z complex. As the proton transferred products were suggested to induce genetic mutations, we propose that the formations of Z(N3 - H)˙ and P(N1 + H)+ in DNA would be mutagenic. These results are expected to help in the understanding of the radiation biology of P and Z in single-stranded and double-stranded DNA.

5-Carboxylcytosine and Cytosine Protonation Distinctly Alter the Stability and Dehybridization Dynamics of the DNA Duplex.

Applications associated with nucleobase protonation events are grounded in their fundamental impact on DNA thermodynamics, structure, and hybridization dynamics. Of the canonical nucleobases, N3 protonation of cytosine (C) is the most widely utilized in both biology and nanotechnology. Naturally occurring C derivatives that shift the N3 pKa introduce an additional level of tunability. The epigenetic nucleobase 5-carboxylcytosine (caC) presents a particularly interesting example since this derivative forms Watson-Crick base pairs of similar stability and displays pH-dependent behavior over the same range as the canonical nucleobase. However, the titratable group in caC corresponds to the exocyclic carboxyl group rather than N3, and the implications of these divergent protonation events toward DNA hybridization thermodynamics, kinetics, and base pairing dynamics remain poorly understood. Here, we study the pH dependence of these physical properties using model oligonucleotides containing C and caC with FTIR and temperature-jump IR spectroscopy. We demonstrate that N3 protonation of C completely disrupts duplex stability, leading to large shifts in the duplex/single-strand equilibrium, a reduction in the cooperativity of melting, and an acceleration in the rate of duplex dissociation. In contrast, while increasing 5-carboxyl protonation in caC-containing duplexes induces an increase in base pair fluctuations, the DNA duplex can tolerate substantial protonation without significant perturbation to the duplex/single-strand equilibrium. However, 5-carboxyl protonation has a large impact on hybridization kinetics by reducing the transition state free energy. Our thermodynamic and kinetic analysis provides new insight on the impact of two divergent protonation mechanisms in naturally occurring nucleobases on the biophysical properties of DNA.

Structures and Relative Glycosidic Bond Stabilities of Protonated 2'-Fluoro-Substituted Purine Nucleosides.

The 2'-substituent is the primary distinguishing feature between DNA and RNA nucleosides. Modifications to this critical position, both naturally occurring and synthetic, can produce biologically valuable nucleoside analogues. The unique properties of fluorine make it particularly interesting and medically useful as a synthetic nucleoside modification. In this work, the effects of 2'-fluoro modification on the protonated gas-phase purine nucleosides are examined using complementary tandem mass spectrometry and computational methods. Direct comparisons are made with previous studies on related nucleosides. Infrared multiple photon dissociation action spectroscopy performed in both the fingerprint and hydrogen-stretching regions allows for the determination of the experimentally populated conformations. The populated conformers of protonated 2'-fluoro-2'-deoxyadenosine, [Adofl+H]+, and 2'-fluoro-2'-deoxyguanosine, [Guofl+H]+, are highly parallel to their respective canonical DNA and RNA counterparts. Both N3 and N1 protonation sites are accessed by [Adofl+H]+, stabilizing syn and anti nucleobase orientations, respectively. N7 protonation and anti nucleobase orientation dominates in [Guofl+H]+. Spectroscopically observable intramolecular hydrogen-bonding interactions with fluorine allow more definitive sugar puckering determinations than possible for the canonical systems. [Adofl+H]+ adopts C2'-endo sugar puckering, whereas [Guofl+H]+ adopts both C2'-endo and C3'-endo sugar puckering. Energy-resolved collision-induced dissociation experiments with survival yield analyses provide relative glycosidic bond stabilities. The N-glycosidic bond stabilities of the protonated 2'-fluoro-substituted purine nucleosides are found to exceed those of their canonical analogues. Further, the N-glycosidic bond stability is found to increase with increasing electronegativity of the 2'-substituent, i.e., H < OH < F. The N-glycosidic bond stability is also greater for the adenine nucleoside analogues than the guanine nucleoside analogues.

Evidence for Hidden Involvement of N3-Protonated Guanine in RNA Structure and Function.

Charged nucleobases have been found to occur in several known RNA molecules and are considered essential for their structure and function. The mechanism of their involvement is however not yet fully understood. Revelation of the role of N7-protonated guanine, in modulating the geometry and stability of noncanonical base pairs formed through its unprotonated edges [Watson–Crick (WC) and sugar], has triggered the need to evaluate the feasibility of similar roles of other protonated nucleobases [Halder et al., Phys Chem Chem Phys, 2015, 17, 26249]. In this context, N3 protonation of guanine makes an interesting case as its influence on the charge distribution of the WC edge is similar to that of N7 protonation, though its thermodynamic cost of protonation is significantly higher. In this work, we have carried out structural bioinformatics analyses and quantum mechanics-based calculations to show that N3 protonation of guanine may take place in a cellular environment, at least in the G:C W:W Trans and G:G W:H Cis base pairs. Our results provide a reasonable starting point for future investigations in order to address the larger mechanistic question.

Gas-phase structures of protonated arabino nucleosides

Nucleoside modification plays an important role in the native function of DNA and RNA and is also an important synthetic tool for pharmaceuticals. The gas-phase structures of several protonated arabino nucleosides, an important family of modified nucleoside pharmaceuticals, are examined in this work. Infrared multiple photon dissociation action spectra are collected for the protonated forms of the adenine, cytosine, guanine, and uracil arabinosides ([araAdo+H]+, [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+) in the IR fingerprint and hydrogen-stretching regions. Electronic structure calculations are performed to determine low-energy conformers of [araAdo+H]+, [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+, and generate predicted IR spectra for comparison to experiments. Conformers displaying a unique O2′H⋯O5′ hydrogen-bonding interaction are populated in each of these systems. Conformers exhibiting hydrogen-bonding interactions between the nucleobase and O5′ are also found to display good agreement with the measured spectra. Competition between sugar–sugar, and nucleobase–sugar hydrogen bonding reveals a preference for [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+ to stabilize the sugar ring pucker over the nucleobase rotation. N3 protonation of [araAdo+H]+ provides a very strong N3H+⋯O5′ hydrogen-bonding interaction, such that nucleobase–sugar hydrogen-bonding takes energetic preference over stabilization of the sugar puckering. However, conformers exhibiting each mode of hydrogen bonding contribute to the measured spectrum of [araAdo+H]+.

Protonation of Nucleobases in Single‐ and Double‐Stranded DNA

Single-stranded model oligodeoxyribonucleotides, each containing a single protonatable base-cytosine, adenine, guanine, or 5-methylcytosine-centrally located in a background of non-protonatable thymine residues, were acid-titrated in aqueous solution, with UV monitoring. The basicity of the central base was shown to depend on the type of the central base and its nearest neighbours and to rise with increasing oligonucleotide length and decreasing ionic strength of the solution. More complex model oligonucleotides, each containing a centrally located 5-methylcytosine base, were comparatively evaluated in single-stranded and double-stranded form, by UV spectroscopy and high-field NMR. The N3 protonation of the 5-methylcytosine moiety in the double-stranded case occurred at much lower pH, at which the duplex was already experiencing general dissociation, than in the single-stranded case. The central guanine:5-methylcytosine base pair remained intact up to this point, possibly due to an unusual alternative protonation on O2 of the 5-methylcytosine moiety, already taking place at neutral or weakly basic pH, as indicated by UV spectroscopy, thus suggesting that 5-methylcytosine sites in double-stranded DNA might be protonated to a significant extent under physiological conditions.

The influence of the metal cations and microhydration on the reaction trajectory of the N3 ↔ O2 thymine proton transfer: Quantum mechanical study.

This study involves the intramolecular proton transfer (PT) process on a thymine nucleobase between N3 and O2 atoms. We explore a mechanism for the PT assisted by hexacoordinated divalent metals cations, namely Mg2+ , Zn2+ , and Hg2+ . Our results point out that this reaction corresponds to a two-stage process. The first involves the PT from one of the aqua ligands toward O2. The implications of this stage are the formation of a hydroxo anion bound to the metal center and a positively charged thymine. To proceed to the second stage, a structural change is needed to allow the negatively charged hydroxo ligand to abstract the N3 proton, which represents the final product of the PT reaction. In the presence of the selected hexaaqua cations, the activation barrier is at most 8 kcal/mol. © 2017 Wiley Periodicals, Inc.

Glutamine Amide Flip Elicits Long Distance Allosteric Responses in the LOV Protein Vivid.

Light-oxygen-voltage (LOV) domains sense blue light through the photochemical formation of a cysteinyl-flavin covalent adduct. Concurrent protonation at the flavin N5 position alters the hydrogen bonding interactions of an invariant Gln residue that has been proposed to flip its amide side chain as a critical step in the propagation of conformational change. Traditional molecular dynamics (MD) and replica-exchange MD (REMD) simulations of the well-characterized LOV protein Vivid (VVD) demonstrate that the Gln182 amide indeed reorients by ∼180° in response to either adduct formation or reduction of the isoalloxazine ring to the neutral semiquinone, both of which involve N5 protonation. Free energy simulations reveal that the relative free energies of the flipped Gln conformation and the flipping barrier are significantly lower in the light-adapted state. The Gln182 flip stabilizes an important hinge-bβ region between the PAS β-sheet and the N-terminal cap helix that in turn destabilizes an N-terminal latch region against the PAS core. Release of the latch, observed both experimentally and in the simulations, is known to mediate light-induced VVD dimerization. This computational study of a LOV protein, unprecedented in its agreement with experiment, provides an atomistic view of long-range allosteric coupling in a photoreceptor.

Computational Assignment of the Histidine Protonation State in (6-4) Photolyase Enzyme and Its Effect on the Protonation Step

The initial step to resolving the controversy regarding the repair mechanism of the pyrimidine (6-4) pyrimidone photoproduct DNA lesion is to determine the protonation state of the two conserved active site histidine residues (His365 and His369 in Drosophila melanogaster) in the (6-4) photolyase enzyme ((6-4) PHR). Both His residues were experimentally determined to be crucial for catalysis. Most previous theoretical studies assumed the presence of protonated His365 in the active site, which would transfer its proton to N3′ of the lesion as the first step in repair; however, other empirical/semiempirical pKa calculations suggested the presence of two neutral His residues in the active site. Here, we conduct molecular dynamics simulations (MD) of the (6-4) PHR/DNA complex on the basis of the X-ray crystal structure to investigate all combinations of the three possible protonation states of each His. The MD results show that HIP365 (both ND1 and NE2 protonated) and HID369 (only ND1 protonated) are the most probable protonation states in the active site. Furthermore, we employ quantum mechanics-cluster and quantum mechanical/molecular mechanical (QM/MM) approaches to investigate the protonation of N3′, starting with three plausible complexes resulting from MD (HIP365/HID369, HID365/HIE369, and HID365/HIP369). Surprisingly, protonation of the N3′ atom is found to be feasible starting from all three protonation states: protonated HIP365 transfers the proton via a barrierless step, and in the other cases of neutral HID365, the proton is transferred from O4′ of the lesion via His365 through an energy barrier of ∼80 kJ mol–1 in both complexes. These results might explain the conservation of repair activity over a wide range of pH values.

Crystal structure of a poly(rA) staggered zipper at acidic pH: evidence that adenine N1 protonation mediates parallel double helix formation

We have solved at 1.07 Å resolution the X-ray crystal structure of a polyriboadenylic acid (poly(rA)) parallel and continuous double helix. Fifty-nine years ago, double helices of poly(rA) were first proposed to form at acidic pH. Here, we show that 7-mer oligo(rA), i.e. rA7, hybridizes and overlaps in all registers at pH 3.5 to form stacked double helices that span the crystal. Under these conditions, rA7 forms well-ordered crystals, whereas rA6 forms fragile crystalline-like structures, and rA5, rA8 and rA11 fail to crystallize. Our findings support studies from ∼50 years ago: one showed using spectroscopic methods that duplex formation at pH 4.5 largely starts with rA7 and begins to plateau with rA8; another proposed a so-called ‘staggered zipper’ model in which oligo(rA) strands overlap in multiple registers to extend the helical duplex. While never shown, protonation of adenines at position N1 has been hypothesized to be critical for helix formation. Bond angles in our structure suggest that N1 is protonated on the adenines of every other rAMP−rAMP helix base pair. Our data offer new insights into poly(rA) duplex formation that may be useful in developing a pH sensor.

N3 Protonation Induces Base Rotation of 2′-Deoxyadenosine-5′-monophosphate and Adenosine-5′-monophosphate

Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments combined with theoretical calculations are performed to investigate the stable gas-phase conformations of the protonated adenine mononucleotides, [pdAdo+H](+) and [pAdo+H](+). Conformations that are present in the experiments are elucidated via comparative analyses of the experimental IRMPD spectra and the B3LYP/6-311+G(d,p) IR spectra predicted for the conformers optimized at this level of theory. N3 protonation is preferred as it induces base rotation, which allows a strong hydrogen bond to be formed between the excess proton of adenine and the phosphate moiety. In contrast, both N1 and N7 protonation are predicted to be >35 kJ/mol less favorable than N3 protonation. Only N3 protonated conformers are present in the experiments in measurable abundance. Both the low-energy conformers computed and the experimental IRMPD spectra of [pdAdo+H](+) and [pAdo+H](+) indicate that the 2'-hydroxyl moiety does not significantly impact the structure of the most stable conformer or the IRMPD spectral profile of [pAdo+H](+) vs that of [pdAdo+H](+). However, the 2'-hydroxyl leads to a 3-fold enhancement in the IRMPD yield of [pAdo+H](+) in the fingerprint region. Comparison of present results to those reported in a previous IRMPD study of the analogous protonated adenine nucleosides allows the effects of the phosphate moiety on the gas-phase conformations to be elucidated.

A Novel Signal Transduction Mechanism in LOV Domain Proteins

In light-oxygen-voltage (LOV) domain photoreceptors, blue light irradiation leads to thioether bond formation between the C4a carbon of the flavin-cofactor isoalloxazine ring and a conserved cysteine residue. Adduct formation results in N5 nitrogen protonation and changes in the hydrogen bond network surrounding the cofactor, instigating protein conformational changes and downstream signaling. Interestingly, when the circadian-clock LOV-protein Vivid from Neurospora crassa is devoid of the adduct-forming cysteine, the neutral semiquinone forms upon photoreduction of the cofactor. This variant is surprisingly still capable of dimerization and in vivo signaling. Moreover, analogous LOV domains of natural cysteine-less proteins were discovered to exhibit conformational changes after either chemical reduction or photoreduction. As flavin N5 protonation is a shared event in both the formations of the adduct and the neutral semiquinone, our findings indicate that 1) the neutral semiquinone is a biologically functional state in LOV-proteins and 2) flavin N5 protonation is sufficient for triggering signal transduction.

Protonation induces base rotation of purine nucleotides pdGuo and pGuo.

Infrared multiple photon dissociation (IRMPD) action spectra of the protonated forms of 2'-deoxyguanosine-5'-monophosphate and guanosine-5'-monophosphate, [pdGuo+H](+) and [pGuo+H](+), are measured over the IR fingerprint and hydrogen-stretching regions using the FELIX free electron laser and an OPO/OPA laser system. Electronic structure calculations are performed to generate low-energy conformations of [pdGuo+H](+) and [pGuo+H](+) and determine their relative stabilities at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparative analyses of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers are performed to determine the most favorable site of protonation and the conformers present in the experiments. These comparisons and the computed energetics find that N7 protonation is considerably preferred over O6 and N3, and the N7 protonated ground-state conformers of [pdGuo+H](+) and [pGuo+H](+) are populated in the experiments. The 2'-hydroxyl substituent does not significantly impact the stable low-energy conformers of [pdGuo+H](+)vs. those of [pGuo+H](+). The effect of the 2'-hydroxyl substituent is primarily reflected in the relative intensities of the measured IRMPD bands, as the IRMPD profiles of [pdGuo+H](+) and [pGuo+H](+) are quite similar. Comparisons to previous IRMPD spectroscopy investigations of the protonated forms of the guanine nucleosides, [dGuo+H](+) and [Guo+H](+), and deprotonated forms of the guanine nucleotides, [pdGuo-H](-) and [pGuo-H](-), provide insight into the effects of the phosphate moiety and protonation on the conformational features of the nucleobase and sugar moieties. Protonation is found to induce base rotation of the guanine residue to an anti orientation vs. the syn orientation found for the deprotonated forms of the guanine nucleotides.