Orientation of a key glutamine residue in the BLUF domain from AppA revealed by mutagenesis, spectroscopy, and quantum chemical calculations

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. A crystal structure of the BLUF domain from AppA suggested that a conserved Gln63 forms a hydrogen bond with the flavin N5 atom. Upon light excitation, this residue is proposed to undergo a approximately 180 degrees rotation that leads to a rearrangement of a hydrogen bonding network. However, crystallographic studies on the other BLUF proteins claimed an opposite orientation for the glutamine residue. In this communication, we have revealed the presence of a Gln63-to-N5 hydrogen bond in the dark state of AppA by a combined approach of mutagenesis, spectroscopy, and quantum chemical calculations. The present finding supports the view that the reorientation of the Gln63 side chain is a key event in the signaling state formation of BLUF proteins.     

Light-induced flipping of a conserved glutamine sidechain and its orientation in the AppA BLUF domain

The AppA BLUF domain is a blue light photoreceptor containing flavin. Conserved glutamine 63 is necessary for the photocycle of the protein, and its side chain has been proposed to flip in response to blue light illumination. Recently published crystal structures of AppA WT and the AppA mutant C20S describe contradictory conclusions regarding the orientation of the conserved glutamine 63 side chain in the dark. Here, we present evidence from NMR spectroscopy confirming light-induced flipping of the glutamine side chain to form a strong hydrogen bond between the glutamine 63 side chain carbonyl group and the tyrosine 21 side chain hydroxyl proton in the light-induced state. Our conclusions are consistent with published data from UV/vis absorbance and FTIR spectroscopy, as well as the crystal structure of AppA WT.     

Factors influencing solvent adduct formation by calixarenes in the solid state

Structural studies of seven very differently functionalised derivatives of calix[4]arene have been used to provide an analysis of the numerous factors which may influence solvent adduct formation by calixarenes. Evidence is presented that even where a solvent guest is included within the calixarene cavity, interactions solely within the cavity cannot be seen as the sole influences upon the guest position and orientation.     

Mass spectrometric investigation of adduct formation by methylated cyclodextrins

FABMS has been employed to detect the formation of supramolecular adducts between methylated cyclodextrins and (i) organic ammonium salts, (ii) organometallic cations, and (iii) neutral metalloorganic complexes. This is a facile technique for studying the second sphere coordination of transition metal complexes by chemically-modified cyclodextrins.     

Energetics and role of the hydrophobic interaction during photoreaction of the BLUF domain of AppA

A recently developed method for time-resolved thermodynamic measurements was used to study the photochemical reaction(s) of the BLUF domain of AppA (AppA-BLUF), which has a dimeric form in the ground state, in terms of the energetics and heat capacity changes (DeltaC(p)) in different time domains. The enthalpy change (DeltaH) of the first intermediate that forms within 1 ns after photoexcitation was 38 (+/-8) kJ mol(-1) at 298 K. The heat capacity change (DeltaC(p)) upon formation of this intermediate was positive [1.4 (+/-0.3) kJ mol(-1) K(-1)]. This positive DeltaC(p) suggests that the hydrophobic surface area of AppA-BLUF exposed to the bulk solvent increased. After this initial transition, a dimerization reaction with another ground-state dimer (i.e., tetramer formation) takes place. Upon this reaction, the energy was stabilized to 26 (+/-6) kJ mol(-1) at 298 K. Interestingly, the dimer formation was accompanied by a larger but negative DeltaC(p) [-6.0 (+/-1) kJ mol(-1) K(-1)]. This negative DeltaC(p) might indicate buried hydrophobic residues at the interface of the dimer and/or the existence of trapped water at the interface. We suggest that hydrophobic interactions are the main driving force for the formation of the dimer upon photoactivation of AppA-BLUF.     

Glutathione traps formaldehyde by formation of a bicyclo[4.4.1]undecane adduct

Glutathione forms complex reaction products with formaldehyde, which can be further modified through enzymatic modification. We studied the non-enzymatic reaction between glutathione and formaldehyde and identified a bicyclic complex containing two equivalents of formaldehyde and one glutathione molecule by protein X-ray crystallography (PDB accession number 2PFG). We have also used (1)H, (13)C and 2D NMR spectroscopy to confirm the structure of this unusual adduct. The key feature of this adduct is the involvement of the gamma-glutamyl alpha-amine and the Cys thiol in the formation of the bicyclic ring structure. These findings suggest that the structure of GSH allows for bi-dentate masking of the reactivity of formaldehyde. As this species predominates at near physiological pH values, we suggest this adduct may have biological significance.     

Sodium adduct formation efficiency in ESI source

Formation of sodium adducts in electrospray (ESI) has been known for long time, but has not been used extensively in practice, and several important aspects of Na⁺ adduct formation in ESI source have been almost unexplored: the ionization efficiency of different molecules via Na⁺ adduct formation, its dependence on molecular structure and Na⁺ ion concentration in solution, fragmentation behaviour of the adducts as well as the ruggedness (a prerequisite for wider practical use) of ionization via Na⁺ adduct formation. In this work, we have developed a parameter describing sodium adducts formation efficiency (SAFE) of neutral molecules and have built a SAFE scale that ranges for over four orders of magnitude and contains 19 compounds. In general, oxygen bases have higher efficiency of Na⁺ adducts formation than nitrogen bases because of the higher partial negative charge on oxygen atoms and competition from protonation in the case of nitrogen bases. Chelating ability strongly increases the Na⁺ adduct formation efficiency. We show that not only protonation but also Na⁺ adduct formation is a quantitative and reproducible process if relative measurements are performed. Copyright © 2013 John Wiley & Sons, Ltd.     

Dichlorosilane-dimethyl ether aggregation: a new motif in halosilane adduct formation

The crystal structures of (H(3)C)(2)O, H(2)SiCl(2) and an adduct of these were determined by low-temperature X-ray crystallography on crystals grown in situ at low temperatures on a diffractometer. The adduct of (H(3)C)(2)O and H(2)SiCl(2) has the composition [(H(3)C)(2)O.H(2)SiCl(2)](2) and contains a four-membered Si(2)O(2) ring, with the Cl atoms pointing to the outside and the Si-H functions pointing to the inner side of the ring. The Si(2)O(2) ring has two longer and two shorter SiO bonds and thus deviates from a square. Quantum chemical calculations give a geometry for [(H(3)C)(2)O.H(2)SiCl(2)](2) which has D(2h) symmetry and allow to obtain an estimate for the adduct formation energies, which are -13.4 kJ mol(-1) for the formation of the mono adduct [(H(3)C)(2)O + H(2)SiCl(2)-->(H(3)C)(2)O.H(2)SiCl(2)], -14.4 kJ mol(-1) for the dimerization of two mono adducts [(H(3)C)(2)O.H(2)SiCl(2)-->[(H(3)C)(2)O.H(2)SiCl(2)](2)] and -41.2 kJ mol(-1) for the reaction 2 (H(3)C)(2)O + 2 H(2)SiCl(2)-->[(H(3)C)(2)O.H(2)SiCl(2)](2). The results are used to rationalize the strongly reduced reactivity of H(2)SiCl(2) towards nucleophilic substitution reactions in (H(3)C)(2)O at low temperatures.     

From crystallographic data to the solution structure of photoreceptors: the case of the AppA BLUF domain

Photoreceptor proteins bind a chromophore, which, upon light absorption, modifies its geometry or its interactions with the protein, finally inducing the structural change needed to switch the protein from an inactive to an active or signaling state. In the Blue Light-Using Flavin (BLUF) family of photoreceptors, the chromophore is a flavin and the changes have been connected with a rearrangement of the hydrogen bond network around it on the basis of spectroscopic changes measured for the dark-to-light conversion. However, the exact conformational change triggered by the photoexcitation is still elusive mainly because a clear consensus on the identity not only of the light activated state but also of the dark one has not been achieved. Here, we present an integrated investigation that combines microsecond MD simulations starting from the two conflicting crystal structures available for the AppA BLUF domain with calculations of NMR, IR and UV-Vis spectra using a polarizable QM/MM approach. Thanks to such a combined analysis of the three different spectroscopic responses, a robust characterization of the structure of the dark state in solution is given together with the uncovering of important flaws of the most popular molecular mechanisms present in the literature for the dark-to-light activation.

With an integrated molecular dynamics and QM/MM strategy we characterize the dark-state structure of a BLUF photoreceptor and ration alize the discrepancy between published crystal structures.     

Ionization of anabolic steroids by adduct formation in liquid chromatography electrospray mass spectrometry

The ionization of 46 anabolic steroids has been studied. The absence of basic or acidic moieties in most of these analytes makes their direct ionization as [M + H]+ by atmospheric pressure interfaces difficult. The formation of adducts with different components of the mobile phase has been found to be an efficient way to ionize anabolic steroids by electrospray. Different mobile phases using methanol (MeOH) or acetonitrile as organic solvent and HCOOH, Na+ or NH4+ as additives have been tested to favor the adduct formation. A direct correlation between the chemical structure of the anabolic steroid and the possibility to ionize it in a particular chromatographic condition has been found. According to their ionization, anabolic steroids can be divided into seven different groups depending on both the nature and the relative position of their functional groups. The formation of different adducts such as [M + Na + MeOH]+ or [M + H + CH3 CN - H2O]+ is required in order to ionize some of these groups and the optimal mobile phase composition for each group of anabolic steroids is proposed. Despite the ionization limitations due to their chemical structure, most of tested anabolic steroids could be ionized using the adduct formation approach.     

Binary adduct formation of desipramine with dicarboxylic acids

This paper presents the results obtained in a screening study for binary adduct formation of desipramine with three dicarboxylic acids—namely succinic, malonic and glutaric acids. Adduct formation is important because most tricyclic antidepressants show limited water solubility as free bases. Three binary adducts were prepared using a wet kneading method in non-homogenous media in presence of ethanol. Investigations of the solid state (ATR-FTIR, PXRD) and thermal analysis (TG/DTG/HF) were used for characterization of the prepared samples. The corroboration of data from employed instrumental techniques suggests that binary adducts in molar ratio 1:1 were obtained under the used experimental conditions.     

Quantifying homo- and heteromolecular hydrogen bonds as a guide for adduct formation

An investigation into the predictability of molecular adduct formation is presented by using the approach of hydrogen bond propensity. Along with the predictions, crystallisation reactions (1a-1j) were carried out between the anti-malarial drug pyrimethamine (1) and the acids oxalic (a), malonic (b), acetylenedicarboxylic (c), adipic (d), pimelic (e), suberic (f), azelaic acids (g), as well as hexachlorobenzene (h), 1,4-diiodobenzene (i), and 1,4-diiodotetrafluorobenzene (j); seven (1a to 1g) of these successfully formed salts. Five of these seven salts were found to be either hydrated or solvated. Hydrogen bond propensity calculations predict that hydrogen bonds between 1 and acids a-g are more likely to form rather than the H bonds involved in self-association, providing a rationale for the observation of the seven new salts. In contrast, propensity of hydrogen bonds between 1 and h-j is much smaller as compared to other bonds predicted for self-association/solvate formation, in agreement with the observed unsuccessful reactions.     

Nanozipper formation in the solid state from a self-assembling tripeptide with a single tryptophan residue

The self-assembly of a terminally protected tripeptide Boc-γ-Abu(1)-Ala(2)-Trp(3)-OMe (γ-Abu = γ-aminobutyric acid) 1 results in the formation of a nanostructured supramolecular zipper through various non-covalent interactions in the crystal in which the indole side-chain of the Trp(3) residue plays a key role via N-H?π interactions.     

Characterization of local polarity and structure of Cys121 domain in beta-lactoglobulin with a new thiol-specific fluorescent probe

The design and synthesis of a new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-maleimidylphenoxyl-1,3,5-triazinylamino)-7-dimethylamino-2-methylphenazine, is reported for characterizing the local polarity and structure, such as the thiol domain, of a protein. The probe comprises a polarity-sensitive fluorophore (neutral red moiety) and a thiol-specific labeling group (maleimidyl moiety). The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity, but not to pH and temperature, which makes the probe suitable for determining the local polarity change of a protein denatured by pH or temperature. The application of this kind has been first demonstrated for the polarity detection of the Cys121 domain of beta-lactoglobulin. It is found that the polarity of the Cys121 domain corresponds to a dielectric constant of 17.3, and this value hardly alters after heat treatment, which may be attributed to the improved thermal reversibility by the Cys121 modification. The simple mixture of the probe and the protein at pH 5.6 is also studied, revealing that the free probe prefers to bind to an outer hydrophobic region. Heat treatment of the mixture causes the modification of Cys121 residues; this modification does not completely destroy the calyx but results in the opening of a channel for the probe to enter the calyx of beta-lactoglobulin. These results show that Cys121 plays an important role not only in the thermal reversibility but also in the accessibility of the calyx to a ligand. The strategy presented here further indicates that the combination of polarity-sensitive fluorescence probe with site-specific labeling may serve as a powerful means for elucidating structures and properties of proteins.     

Sodium 2-mercaptoethanesulfonate in reversible adduct formation and water solubilization

Sodium 2-mercaptoethanesulfonate (MESNA, coenzyme M) forms 1:1 covalent adducts with highly pi-electron deficient heterocycles. The addition is caused by the thiol function, and the adducts become water soluble as sulfonates. 1H NMR spectroscopy has been used to obtain information about electronic and steric effects on the equilibria between 2-pyrimidinones and their 1:1 MESNA adducts. The adducts are potential prodrugs for biologically interesting 2-pyrimidinones.     

Enhancing the formation and capacitance properties of interfacial polymerized polyaniline nanofibers by introducing small alcohol molecules

Journal of Solid State Electrochemistry - Three kinds of polyaniline (PANI) nanofibers were prepared by introducing small alcohol molecules including methanol and ethanol as co-solvents into...     

Thermodynamics and kinetics of methylboroxine.amine adduct formation: a computational study

Density functional theory (B3LYP//6-311+G*) calculations including Poisson-Boltzmann implicit solvent were applied to study the formation of the trimethylboroxine.amine adduct with respect to methylboronic acid monomers and free amine in solution. Potential intermediates and transition states between intermediates were calculated to assess the thermodynamic and kinetic factors controlling this transformation. Our calculations suggest that the rate-determining steps are condensation reactions to form dimers and trimers, and closure of the boroxine ring. Fast amine exchange is expected throughout the transformation, and the most-stable intermediate is a dimer.amine adduct. Using our calculated barriers for the methyl system as a template, we assess the conversion of phenylboronic acid to the triphenylboroxine.amine adduct and find that the pathway is most likely similar, except that the transformation is thermodynamically and kinetically more favored for the phenyl system in the presence of pyridine.