Gallium nanoparticles as novel inhibitors of Aß40 aggregation.

Alzheimer's disease (AD) has been consistently related to the formation of senile amyloid plaques mainly composed of amyloid ß (Aß) peptides. The toxicity of Aß aggregates has been indicated to be responsible for AD pathology. One scenario to decrease Aß toxicity is the development of effective inhibitors against Aß amyloid formation. In this study, we investigate the effect of gallium nitride nanoparticles (GaN NPs) as inhibitors of Aß40 amyloid formation using a combination of biophysical approaches. Our results show that the lag phase of Aß40 aggregation kinetics is significantly retarded by GaN NPs in a concentration dependent manner, implying the activity of GaN NPs in interfering with the formation of the crucial nucleus during Aß aggregation. Our results also show that GaN NPs can reduce the amyloid fibril elongation rate in the course of the aggregation kinetics. It is speculated that the high polarization characteristics of GaN NPs may provoke a strong interaction between the particles and Aß40 peptide and in this way decrease self-association of the peptide monomers to form amyloids.

In situ analysis and imaging of aromatic amidine at varying ligand densities in solid phase.

We report the development of a fast and accurate fluorescence-based assay for amidine linked to cellulose membranes and Sepharose gel. The assay is founded on the glyoxal reaction, which involves reaction of an amidine group with glyoxal and an aromatic aldehyde, leading to the formation of a fluorophore that can be analyzed and quantified by fluorescence spectroscopy and imaging. While the assay has been reported previously for aromatic amidine estimation in solution phase, here we describe its adaptation and application to amidine linked to diverse forms of solid matrices, particularly benzamidine Sepharose and benzamidine-linked cellulose membranes. These functionalized porous matrices find important application in purification of serine proteases. The efficacy of a protein separation device is determined by, among other factors, the ligand (amidine) density. Hence, a sensitive and reproducible method for amidine quantitation in solid phase is needed. The glyoxal reaction was carried out on microbead-sized Sepharose gel and cellulose membranes. Calibration curves were developed for each phase, which established linearity in the range of 0-0.45 µmol per mL amidine for free amidine in solution, 0-0.45 µmol amidine per mL Sepharose gel, and 0-0.48 µmol per mL cellulose membrane. The assay showed high accuracy (~ 3.4% error), precision (RSD < 2%), and reproducibility. Finally, we show how this fluorescent labeling (glyoxal) method can provide a tool for imaging membranes and ligand distribution through confocal laser scanning microscopy. Graphical abstract.

Site-specific dynamics of amyloid formation and fibrillar configuration of Aß(1-23) using an unnatural amino acid.

We identify distinct site-specific dynamics over the time course of Aß1-23 amyloid formation by using an unnatural amino acid, p-cyanophenylalanine, as a sensitive fluorescent and Raman probe. Our results also suggest the key role of an edge-to-face aromatic interaction in the conformational conversion to form and stabilize ß-sheet structure.

Aggregation gatekeeper and controlled assembly of Trpzip ß-hairpins.

Protein and peptide aggregation is an important issue both in vivo and in vitro. Herein, we examine the aggregation behaviors of two well-studied ß-hairpins, Trpzip1 and Trpzip2. Previous studies suggested that Trpzip2 remains monomeric up to a concentration of ~15 mM whereas Trpzip1 readily aggregates at micromolar concentrations at acidic or neutral pH. This disparity is puzzling considering that these two peptides differ only in their turn sequences (i.e., GN vs NG). We hypothesize that these peptides can aggregate from their folded states via native edge-to-edge interactions and that the Lys8 residue in Trpzip2 is a more effective aggregation gatekeeper, because of a more favorable orientation. In support of this hypothesis, we find that increasing the pH to 13 or replacing Lys8 with a hydrophobic and photolabile Lys analogue, Lys(nvoc), leads to a significant increase in the aggregation propensity of Trpzip2, and that the aggregation of this Trpzip2 mutant can be reversed upon restoring the native Lys side chain via photocleavage of the nvoc moiety. In addition, we find that while both Trpzip1 and Trpzip2 form parallel ß-sheet aggregates, the Lys(nvoc) Trpzip2 mutant forms antiparallel ß-sheets and more stable fibrils. Taken together, these findings provide another example showing how sensitive peptide and protein aggregation is to minor sequence variation and that it is possible to use a photolabile non-natural amino acid, such as Lys(nvoc), to tune the rate of peptide aggregation and to control fibrillar structure.

Photooxidation mechanism of levomepromazine in different solvents.

Unwanted photoinduced responses are well-known adverse effects of most promazine drugs, including levomepromazine (LPZ, Levoprome(®) or Nozinan(®)). This drug is indicated in psychiatry primarily for the treatment of schizophrenia and other schizoaffective disorders. Levomepromazine's particular sedative properties make it especially fit for use in psychiatric intensive care. Nevertheless, it is photolabile under UV-A and UV-B light in aerobic conditions resulting in the formation of its sulfoxide. The LPZ photochemistry in acetonitrile (MeCN) is completely different from that in methanol (MeOH) and phosphate buffer solutions (PBS, pH = 7.4). The major photoproduct in PBS and MeOH under aerobic conditions is levomepromazine sulfoxide (LPZSO), although the amount is considerably higher in the aqueous environment. The corresponding main photoproduct in MeCN could not be characterized. The destruction quantum yields of LPZ in PBS, MeOH and MeCN are 0.13, 0.02 and <10(-3), respectively. It is further demonstrated that LPZSO does not form by the reaction of singlet oxygen with ground-state LPZ. This oxidation product is actually produced by the reaction of the cation radical of LPZ (LPZ·(+)) with molecular oxygen. This cation radical in turn, is produced by an electron transfer process between the (3) LPZ* and ground-state molecular oxygen.

Infrared study of the stability and folding kinetics of a series of ß-hairpin peptides with a common NPDG turn.

The thermal stability and folding kinetics of a series of 15-residue ß-hairpins with a common Type I [3:5] NPDG turn were studied using Fourier transform infrared spectroscopy (FTIR) and laser-induced temperature jump (T-jump) with infrared detection, respectively. Mutations at positions 3, 5, or 13 in the peptide sequence SEXYXNPDGTWTXTE, where X represents the position of mutation, were performed to study the roles of hydrophobic interactions in determining the thermodynamic and kinetic properties of ß-hairpin folding. The thermal stability studies show a broad thermal folding/unfolding transition for all the peptides. T-jump studies indicate that these ß-hairpin peptides fold in less than 2 µs. In addition, both folding and unfolding rate constants decrease with increasing strength of hydrophobic interactions. Kinetically, the hydrophobic interactions have more significant influence on the unfolding rate than the folding rate. Φ-value analysis indicates that the hydrophobic interactions between the side chains are mainly formed at the latter part of the transition-state region during the folding process. In summary, the results suggest that the formation of the native structure of these ß-hairpins depends on the correct topology of the hydrophobic cluster. Besides the formation of the turn region as a key process for folding as suggested by previous studies, a hydrophobic collapse process may also play a crucial role during ß-hairpin folding.

Solvent dependence of the photophysical properties of 2-chlorothioxanthone, the principal photoproduct of chlorprothixene.

2-chlorothioxanthone (CTX) is used as photoinitiator for the reticulation of synthetic resins and for the preparation of pharmaceuticals. It was previously determined that CTX is the primary photoproduct of z-chlorprothixene (CPTX) when irradiated at 313 nm and is formed in an autocatalyzed reaction through an energy-transfer mechanism (Piñero et al. [2009] Photochem. Photobiol., 85, 895-900). In this work, the photophysical properties of CTX were measured in acetonitrile/water solutions to determine if their magnitude can affect the side effects of CPTX. The results show that CTX has higher absorption coefficients in the visible region (400-420 nm) and higher triplet quantum yields than its parent compound. Similar to TX, both properties strongly depend on the solvent polarity/hydroxylicity. The quantum yield of the triplet intermediate is very close to the value of the phenothiazine triplets. The phenothiazines are the most phototoxic antidepressants. Therefore, given the appropriate microenvironment, the photosensitization side effects of CPTX can be intensified on the production of CTX.

Photophysics and photochemistry of z-chlorprothixene in acetonitrile.

Chlorprothixene (CPTX, Taractan) is a low potency antipsychotic mainly used for the treatment of psychotic disorders (e.g. schizophrenia) and acute mania occurring as part of bipolar disorders. As in the case of other numerous drugs used in the treatment of psychiatric disorders, CPTX presents geometric isomerism. Therefore, in vitro irradiation induces a rapid Z/E isomerization, which can affect its pharmacokinetic properties. This photoisomerization is not dependent on the oxygen concentration. The Z/E quantum yields determined for zCPTX in acetonitrile are 0.22 and 0.21 in anaerobic and aerobic environments, respectively. In the presence of water, both isomers decompose to produce 2-chlorothioxanthone (CTX) after prolonged irradiation. This process strongly depends on the water concentration and the irradiation time, i.e. it is autocatalyzed by the CTX through a triplet-energy transfer mechanism. The protonation state of the terminal amino group, on the other hand, has no effect on the isomerization process, but inhibits the formation of CTX. These results indicate that the phototoxicity of zCPTX is somehow affected by the formation of CTX.

Photodegradation of 2-chloro substituted phenothiazines in alcohols.

The mechanisms that trigger the phototoxic response to 2-chlorophenothiazine derivatives are still unknown. To better understand the relationship between the molecular structure of halogenated phenothiazines and their phototoxic activity, their photophysics and photochemistry were studied in several alcohols. The photodestruction quantum yields were determined under anaerobic conditions using monochromatic light (313 nm). Absorption- and emission-spectroscopy, (1)H- and (13)C-NMR and GC-MS were used to characterize the photoproducts and reference compounds. An electron transfer mechanism had been previously proposed by Bunce et al. (J. Med. Chem. 22, 202-204) to explain the large difference between the photodestruction quantum yield of 2-chlorpromazine (phi = 0.46) and 2-chlorphenothiazine (phi = 0.20). According to these authors, the alkylamino chain transfers an electron to the phenothiazine moiety. Our results demonstrate that this mechanism is incorrect, because the photodestruction quantum yields of all chlorinated derivatives of this study are the same under the same conditions of solvent and irradiation wavelength. The quantum yield has no dependence on the 10-substituent, but it depends on the solvent. The percentage of each photoproduct, on the other hand, strongly depends on that substituent, but not very much on the solvent. Finally, it is demonstrated that the phototoxic effect of chlorinated phenothiazines is not related to the photodechlorination, although both processes share the same transient.

SPECTROSCOPIC AND ELECTROCHEMICAL PROPERTIES OF 2-AMINOPHENOTHIAZINE.

Phenothiazines derivatives are versatile compounds that are used in many fields, depending on the type and position of the substitution on the parent molecule. The photochemical, photophysical and electrochemical properties of several phenothiazine derivatives have been previously reported in detail. However, no reports have been presented for 2-aminophenothiazine (APH), a candidate that provides for the further chemical modification and the introduction of specific substituents. In this work, the photophysical and electrochemical properties of APH were measured in acetonitrile. The APH ground state absorption and fluorescence spectrum (phi(f) < 0.01) are similar to the corresponding that of PH parent molecule. A mono exponential decay fluorescence lifetime of 0.65 ns was determined for APH in acetonitrile. Characterization of the 355 nm nanosecond laser flash photolysis transient species reveals the presence of the triplet-triplet transient intermediate with a high intersystem crossing quantum yield (phi(T) = 0.72 +/- 0.07), indicating that the APH main excited state deactivation channel is intersystem crossing. The oxidation potential of APH is lower than phenothiazine parent molecule ((0.38 V vs 0.69 V vs Ag/AgCl(sat)). Altogether, these results show that APH has photochemical and photophysical properties similar to the phenothiazine parent molecule, but with the possibility of providing an amino functionality at 2-position for further chemical modification.

Photophysics and photochemistry of imipramine, desimipramine, and clomipramine in several solvents: a fluorescence, 266 nm laser flash, and theoretical study.

Imipramine (IPA) and its derivatives are used widely for the treatment of depression and other mental disorders. Although there are more than 20 FDA-approved antidepressant drugs, the search continues for better compounds with fewer deleterious side effects and higher efficacy. Over the past decade, several classes of antipsychotic drugs have been developed, which-in spite of their structural diversity-share an ability to modulate neurotransmission and to produce undesirable side effects. Phototoxicity is one of the most important side effects noted in treatment with tricyclic antidepressants (TCAs), but its mechanism has not yet been elucidated. To develop new knowledge regarding the relationship between the structure and the photophysics of these TCAs, we measured the photophysical properties of IPA, desimipramine (DIPA), and clomipramine (CIPA) in different solvents. The electronic configurations for the ground and the first excited singlet states were calculated using the AM1/RHF/CI and the AM1/RHF/HE semiempirical quantum theoretical methods, respectively. The ground-state properties are solvent-independent, while the emission maxima are red-shifted with increasing solvent polarity/polarizability. However, the fluorescence quantum yield is relatively low in all of the tested solvents (phif<0.02). The primary transient intermediates produced by 266 nm high-intensity laser photolysis are the solvated electron and the corresponding radical cation, with a negligible contribution of triplet-triplet absorption. The properties determined for the primary transients generated with a 266 nm laser flash are consistent with the photodamaging effects generated through a limited radical mechanism.

Role of sequence and conformation on the photochemistry and the photophysics of A-T DNA dimers: an experimental and computational approach.

The role of base sequence and conformation on the photochemistry and photophysics of thymidylyl (3'-5')-2'-deoxyadenosine sodium salt (TpdA) and 2-deoxyadenylyl (3'-5')-thymidine ammonium salt (dApT) was studied. To this end, nanosecond transient absorption at 266 nm, steady-state irradiation at 254 nm, and quantum chemical calculations were used. The transient absorption spectra show the solvated electron broad band in the visible region for each dinucleotide. In addition, low-intensity absorption bands are observed in the UV region, which are attributed to the deprotonated and protonated neutral radicals of adenine and thymine bases. Photoionization (PI) occurs by one- and two-photon pathways; the latter accounting for approximately 70% of the net PI yield. A diffusion-limited rate constant of 2.0 x 10(10) M(-1) s(-1) was obtained for the reaction of the neutral molecule with the photoejected electron in both sequences. The photodestruction yield, measured from the chromophore loss at 260 nm, decreases in the presence of well-known electron scavengers. This suggests the participation of base radical anions as one of the photodegradation pathways, which is higher in TpdA than in dApT. The intermediacy of a radical ion pair (charge separated state) between the adjacent adenine and thymine bases is proposed in the formation of the [2 + 2] cycloadduct intermediate. The [2 + 2] cycloadduct intermediate is known to be the precursor of the thymine-adenine eight-member ring photoproduct (TA*). Conformational constrains in the radical ion pair are suggested to explain the absence of the TA* photoproduct in dApT. This hypothesis is supported by semiempirical calculations performed on all relevant reactive intermediates proposed to participate in the mechanism of formation of TA*. Altogether, the results show that sequence and conformation profoundly influence the photochemistry and the photophysics of these DNA model systems.

A novel fluorescent probe for protein binding and folding studies: p-cyano-phenylalanine.

Recently, it is has been shown that the C=N stretching vibration of a non-natural amino acid, p-cyano-phenylalanine (PheCN), could be used as an infrared reporter of local environment. Here, we further showed that the fluorescence emission of PheCN is also sensitive to solvent and, therefore, could be used as a novel optical probe for protein binding and folding studies. Moreover, we found that the fluorescence quantum yield of PheCN is nearly five times larger than that of phenylalanine and, more importantly, can be selectively excited even when other aromatic amino acids are present, thus making it a more versatile fluorophore. To test the feasibility of using PheCN as a practical fluorescent probe, we studied the binding of calmodulin (CaM) to a peptide derived from the CaM-binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK3CN) contains a single PheCN residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK3CN solution resulted in quenching of the PheCN fluorescence. A series of stochiometric titrations further allowed us to determine the binding affinity (Kd) of this peptide to CaM. Taken together, these results indicated that the PheCN fluorescence is sensitive to environment and could be applicable to a wide variety of biological problems.

Alpha 1-antitrypsin polymerization: a fluorescence correlation spectroscopic study.

Alpha(1)-antitrypsin (AT) is the most abundantly circulating human proteinase inhibitor in the serpin family. The polymerization of AT, leading to alpha(1)-antitrypsin deficiency, has been studied extensively in vitro by a variety of ensemble methods. Here we report the use of fluorescence correlation spectroscopy to gain further insight into this process. Measurements of the distributions of diffusion times of polymerizing AT, carried out at 45, 50, and 55 degrees C, clearly show the existence of a kinetic lag phase, during which short oligomers are formed, prior to the formation of heterogeneous mixtures of longer polymers, and suggest that long polymers, which appear to be metastable, are produced through the condensation of shorter oligomers.

Substitution and solvent effects on the photophysical properties of several series of 10-alkylated phenothiazine derivatives.

The photophysical properties of several 2-substituted, 10-alkylated phenothiazines were measured in several solvents to investigate the relevance of the molecular structure in their photophysics and consequent photochemistry. Because the interaction modes of each drug and its corresponding species strongly depend on the variety of microenvironments in the cells, the properties of each one of these species must also be determined separately to understand fully the mechanism of action of the drug and the mechanism of its side effects. Information on the chemical interactions of the different species at the cellular level can be inferred from the corresponding electronic properties. In this work, we present absorption, steady-state, and time-resolved emission, laser flash photolysis, and quantum theoretical results for the ground state, the first excited singlet and triplet states, and the cation radical of promazine hydrochloride (PZ), 2-chlorpromazine hydrochloride (CPZ), 2-trifluoromethylpromazine hydrochloride (TFMPZ), 2-trifluoromethylperazine dihydrochloride (TFMP), 2-thiomethylpromazine (TMPZ), and thioridazine hydrochloride (TR). The corresponding nonalkylated phenothiazines are included as references. The photophysical properties of this drug family depend more on the solvent and the 2-substituents than on the dialkylaminopropyl chain. The largest effect was found for the triplet state of the 2-halogenated derivatives in phosphate buffer (PBS). Both the quantum yield and the lifetime of this intermediate drop to less than 5% of the corresponding value in organic solvents. The triplet state of halogenated promazines is efficiently quenched by a proton-transfer mechanism, and the rate of this quenching correlates very well with the phototoxicity of the promazine drugs. Therefore, we postulate that this species is directly related to the phototoxic side effect of neuroleptic drugs.

Conformational distribution of a 14-residue peptide in solution: a fluorescence resonance energy transfer study.

We demonstrate here that a nitrile-derivatized phenylalanine residue, p-cyanophenylalanine (Phe(CN)), and tryptophan (Trp) constitute a novel donor-acceptor pair for fluorescence resonance energy transfer (FRET). The Förster distance of this FRET pair was determined to be approximately 16 A and hence is well suited for determining relatively short separation distances. To validate the applicability of this FRET pair in conformational studies, we studied the conformational heterogeneity of a 14-residue amphipathic peptide, Mastoparan X (MPx peptide), in water and 7 M urea solution as well as at different temperatures. Specifically, seven nitrile-derivatized mutants of the MPx peptide, each containing a Phe(CN) residue that replaces different positions along the peptide sequence (i.e., from position 5 to 11) and serves as a resonance energy donor to the native Trp residue at position 3, were studied spectroscopically. The FRET efficiencies obtained from these peptides allowed us to gain a global picture regarding the conformational distribution of the MPx peptide in different environments. Our results suggest that the MPx molecules exist in water as an ensemble of rather compact conformations, with a radius of gyration of approximately 4.2 A, whereas in 7 M urea the radius of gyration increases to approximately 6.5 A, indicating that the peptide conformations become more extended under this condition. However, we found that temperature had only a negligible effect on the size of the MPx peptide, underlining the difference between the thermally and chemically denatured states of polypeptides. The application of the Gaussian chain or the wormlike chain model allowed us to further obtain the probability distribution function of the separation distance between any two residues along the peptide sequence. We found that the effective bond length of the MPx peptide, obtained by using the Gaussian chain model, is 2.78 A in water and 4.28 A in 7 M urea.

Infrared study of the stability and folding kinetics of a 15-residue beta-hairpin.

The thermal stability and folding kinetics of a 15-residue beta-hairpin (SESYINPDGTWTVTE) have been studied by using infrared (IR) spectroscopy coupled with laser-induced temperature-jump (T-jump) technique for rapid folding-unfolding initiation. An alternative method based on analyzing IR difference spectra was also introduced to obtain thermodynamic properties of beta-sheets, which complements the commonly used circular dichroism (CD) and fluorescence techniques. Equilibrium IR measurements indicate that the thermal unfolding of this beta-hairpin is fairly broad. However, it can be described by a two-state transition with a thermal melting temperature of approximately 29 degrees C. Time-resolved IR measurements following a T-jump, probed at 1634 cm(-1), indicate that the folding of this beta-hairpin follows first-order kinetics and is amazingly fast. At 300 K, the folding time is approximately 0.8 micros, which is only 2-3 times slower than that of alpha-helix formation. Additionally, the energetic barrier for folding is small (approximately 2 kcal mol(-1)). These results, in conjunction with results from other studies, support a view that the details of native contacts play a dominant role in the kinetics of beta-hairpin folding.