Chemo-optogenetic Protein Translocation System Using a Photoactivatable Self-Localizing Ligand.

Manipulating subcellular protein localization using light is a powerful approach for controlling signaling processes with high spatiotemporal precision. The most widely used strategy for this is based on light-induced protein heterodimerization. The use of small synthetic molecules that can control the localization of target proteins in response to light without the need for a second protein has several advantages. However, such methods have not been well established. Herein, we present a chemo-optogenetic approach for controlling protein localization using a photoactivatable self-localizing ligand (paSL). We developed a paSL that can recruit tag-fused proteins of interest from the cytoplasm to the plasma membrane within seconds upon light illumination. This paSL-induced protein translocation (paSLIPT) is reversible and enables the spatiotemporal control of signaling processes in living cells, even in a local region. paSLIPT can also be used to implement simultaneous optical stimulation and multiplexed imaging of molecular processes in a single cell, offering an attractive and novel chemo-optogenetic platform for interrogating and engineering dynamic cellular functions.

Microwave-assisted cleavage of cysteine perfluoroaryl thioethers.

The cysteine- perfluoroarene SNAr reaction allows for the sequence-specific attachment of dyes and affinity tags to peptides and proteins. However, while many methods exist for the desulfuration of native and functionalized cysteine residues, there are no reports of their application to perfluoroarylated cysteines. Herein we report both the hydrogenolysis of a perfluoroarylated cysteine to alanine and elimination to dehydroalanine, reactions that are both accelerated by microwave irradiation.

Photorelease Dynamics of Nitric Oxide from Cysteine-Bound Roussin's Red Ester.

Nitric oxide (NO) can either boost or impede the growth of cancer cells depending on its concentration. Therefore, any anticancer treatment using NO requires precisely controlled NO administration to the target cells in terms of dosage and timing. In this context, photochemically activated NO donors were actively explored, but their detailed NO-releasing dynamics, which is crucial for their use, is not known yet. We determined detailed photoexcitation dynamics of a stable, nontoxic, and water-soluble NO precursor, cysteine-bound Roussin's Red Ester (Cys-RRE), including secondary reactions of the nascent photoproducts. The primary quantum yields of the NO dissociation from the photoexcited Cys-RRE were found to be 24-54% depending on the excitation wavelength; however, the geminate rebinding of NO with the nascent radical reduced the level of biologically available NO to as low as 12%. Such information is useful to achieve efficient NO delivery to practical chemical and biological targets.

Flavin-Radical Formation in the Light-Oxygen-Voltage-Sensing Domain of the Photozipper Blue-light Sensor Protein.

Formation of the neutral flavin radical in the light-oxygen-voltage-sensing (LOV-sensing) domain of photozipper, based on VfAUREO1, was investigated by electron paramagnetic resonance spectroscopy. The flavin radical was observed in the presence of dithiothreitol by illumination of a LOV-domain mutant (C254S), in which a photoactive cysteine residue in close proximity to flavin was replaced with a serine. The radical did not form under low initial protein-concentration conditions (less than 20 µM). The flavin radicals accumulated with logistic time-dependent kinetics when the protein concentrations were higher than 30 µM. These results indicate that the radical is produced by concerted reactions involving protein interactions and that the radical is formed from the LOV dimer but not the LOV monomer. In contrast, logistic time dependencies were not observed for the sample adapted to the dark following radical formation by illumination, indicating that initialization of the proton pathway is essential for this fast sensing reaction.

Targeted Annotation of S-Sulfonylated Peptides by Selective Infrared Multiphoton Dissociation Mass Spectrometry.

Protein S-sulfinylation (R-SO2-) and S-sulfonylation (R-SO3-) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 µm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site.

Preparation, stability, and photoreactivity of thiolato ruthenium polypyridyl complexes: Can cysteine derivatives protect ruthenium-based anticancer complexes?

Ruthenium polypyridyl complexes may act as light-activatable anticancer prodrugs provided that they are protected by well-coordinated ligands that i) prevent coordination of other biomolecules to the metal center in the dark and ii) can be removed by visible light irradiation. In this paper, the use of monodentate thiol ligands RSH as light-cleavable protecting groups for the ruthenium complex [Ru(tpy)(bpy)(OH2)](PF6)2 ([1](PF6)2; tpy=2,2';6',2″-terpyridine, bpy=2,2'-bypyridine), is investigated. The reaction of [1](2+) with RSH=H2Cys (L-cysteine), H2Acys (N-acetyl-L-cysteine), and HAcysMe (N-acetyl-L-cysteine methyl ester), is studied by UV-visible spectroscopy, NMR spectroscopy, and mass spectrometry. Coordination of the monodentate thiol ligands to the ruthenium complex takes place upon heating to 353 K, but full conversion to the protected complex [Ru(tpy)(bpy)(SR)]PF6 is only possible when a large excess of ligand is used. Isolation and characterization of the two new thiolato complexes [Ru(tpy)(bpy)(κS-HCys)]PF6 ([2]PF6) and [Ru(tpy)(bpy)(κS-HAcys)]PF6 ([3]PF6) is reported. [3]PF6 shows a metal-to-ligand charge-transfer absorption band that is red shifted (λmax=492 nm in water) compared to its methionine analogue [Ru(tpy)(bpy)(κS-HAmet)](Cl)2 ([5](Cl)2, λmax=452 nm; HAmet=N-acetyl-methionine). In the dark the thiolate ligand coordinated to ruthenium is oxidized even by traces of oxygen, which first leads to the sulfenato, sulfinato, and disulfide ruthenium complexes, and finally to the formation of the aqua complex [1](2+). [3]PF6 showed slow photosubstitution of the thiolate ligand by water under blue light irradiation, together with faster photooxidation of the thiolate ligand compared to dark conditions. The use of thiol vs. thioether monodentate ligands is discussed for the protection of anticancer ruthenium-based prodrugs.

Tyrosine/cysteine cluster sensitizing human γD-crystallin to ultraviolet radiation-induced photoaggregation in vitro.

Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein-aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. γD-Crystallin (HγD-Crys) is abundant in the nucleus of the human lens, and its folding and aggregation have been extensively studied. Previous work showed that HγD-Crys photoaggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surprisingly, the tryptophan residues play a photoprotective role because of a distinctive energy-transfer mechanism. HγD-Crys also contains 14 tyrosine residues, 12 of which are organized as six pairs. We investigated the role of the tyrosines of HγD-Crys by replacing pairs with alanines and monitoring photoaggregation using light scattering and SDS-PAGE. Mutating both tyrosines in the Y16/Y28 pair to alanine slowed the formation of light-scattering aggregates. Further mutant studies implicated Y16 as important for photoaggregation. Mass spectrometry revealed that C18, in contact with Y16, is heavily oxidized during UVR exposure. Analysis of multiple mutant proteins by mass spectrometry suggested that Y16 and C18 likely participate in the same photochemical process. The data suggest an initial photoaggregation pathway for HγD-Crys in which excited-state Y16 interacts with C18, initiating radical polymerization.

Effects of gamma-ray-induced free radicals on the metal content and amino acid composition of human metallothionein-1.

Metallothioneins (MTs), a low-mass class of metalloproteins, are characterized by a high thiolate sulphur and metal content. MTs are involved in metal homeostasis and heavy metal detoxification, and are efficient scavengers of free radicals. This article describes zinc release from human MT-1 and modification of its amino acid composition when subjected to free radicals generated during gamma ray radiolysis. The effect of gamma ray radiolysis of untreated and metal-depleted human MT-1 was tested under multiple aerobic and anaerobic conditions at increasing irradiation doses. Under all conditions, a rapid increase of serine in the early stages of irradiation was observed. Irradiation for longer times led to cysteic acid formation, except under argon atmosphere. Several other amino acid concentrations gradually decreased. Formation of limited amounts of hydroxyproline, hydroxylysine and ornithine as well as some less common derivatives such as cystathionine occurred as side-effects.

L-cysteine functionalized gold nanoparticles for the colorimetric detection of Hg2+ induced by ultraviolet light.

A simple, cost-effective yet rapid and sensitive colorimetric sensor for the detection of Hg(2+) using L-cysteine functionalized gold nanoparticles induced by ultraviolet radiation was developed. The sensitivity and selectivity of detection was also investigated. The L-cysteine modified gold nanoparticles can be induced to aggregate quickly in the presence of Hg(2+), especially with the assistance of ultraviolet radiation. The presence of Hg(2+) can be monitored by the colorimetric response of gold nanoparticles. The detection of Hg(2+) could be realized, after measuring the UV-vis spectra, with a detection limit of 100 nM. The selectivity of this method has been investigated by other divalent metal ions. The effective colorimetric sensor can be used for on-site and real-time Hg(2+) detection.

The effective atomic number revisited in the light of modern photon-interaction cross-section databases.

The effective atomic number, Z(eff), has been calculated for fatty acids and cysteine. It is shown that Z(eff) is a useful parameter for low-Z materials at any energy above 1 keV. Absorption edges of medium-Z elements may complicate the energy dependence of Z(eff) below 10 keV. The notion of Z(eff) is perhaps most useful at energies where Compton scattering is dominating, and where Z(eff) is equal to the mean atomic number, Z, over a wide energy range around 1 MeV.

Photoactivity of kynurenine-derived UV filters.

Quantum yields of photodecomposition and triplet state formation under aerobic and anaerobic conditions are determined for kynurenine (KN), 3-hydroxykynurenine (3OHKN), xanthurenic acid (XAN), and kynurenine adducts of glutathione (GSH-KN), cysteine (Cys-KN), histidine (His-KN), and lysine (Lys-KN) in aqueous solutions. The highest yields of anaerobic photodecomposition were obtained for GSH-KN and His-KN adducts, which correlates with the highest triplet yields for these compounds. In aerobic conditions, the photodecomposition yields for all compounds under study increase; the highest decomposition rates were observed for His-KN and 3OHKN. The fast decomposition of the latter is attributed to the dark autoxidation of the starting compound.

Photoaddition of fluphenazine to nucleophiles in peptides and proteins. Possible cause of immune side effects.

By the action of UVA light, fluphenazine reacted with nucleophiles through a mechanism involving defluorination of its trifluoromethyl group, giving rise to carboxylic acid derivatives that were easily detected by electrospray mass spectrometry. This photoreaction took place with alcohols, sulphydryls, and amines. When irradiation of fluphenazine was carried out in the presence of an amino acid at pH 7.4, the alpha-amino group was covalently bound to the drug. With amino acids possessing a further nucleophilic residue on the side chain, such as lysine, tyrosine, and cysteine--but not serine--both groups reacted, resulting in a fluphenazine-amino acid-fluphenazine diadduct. The same occurred with the physiological peptide glutathione (gamma-glutamylcysteinylglycine). By means of MALDI mass spectrometry, it was shown that fluphenazine also covalently bound to peptides and proteins such as calmodulin. This binding may result in the formation of antibodies, ultimately leading to the destruction of the granulocytes and thus suggesting that photoactivation of this drug may play a role in its clinical side effects, such as agranulocytosis.

Photodegradation and in vitro phototoxicity of aceclofenac.

Aceclofenac (Airtal) (1) is a photoallergic and phototoxic anti-inflammatory and analgesic agent. This drug is photolabile under aerobic and anaerobic conditions. Irradiation of an ethanol-solution of aceclofenac under oxygen or argon at 290-320 nm affords via decarboxlation compound 2 as the main isolated and spectroscopically identified photoproduct, besides hydroxylamine derivates 3 and 4. A radical intermediate was evidenced spectrophotometrically with GSH and DTNB, as well as by the dimerization of cysteine. Red blood cell lysis photosensitized by 1-4 was investigated. Furthermore, in a lipid-photoperoxidation test with linoleic acid the in vitro phototoxicity of aceclofenac was also verified. The photoinduced generation of peroxide by compound 1 was determined during the irradiation in presence of NADPH by chemiluminescence. In relation to the photoallergic activity of this drug, the interaction of aceclofenac with human serum albumin (HSA) has been studied through fluorescence spectroscopy. No photoinduced binding was observed after irradiation of compounds 1 in the presence of human serum albumin.

Photofragmentation of C,N-protected alpha-amino acids: comparing tert-leucine with sulfur-containing amino acids methionine and cysteine.

The photochemical fingerprint for the N-acetyl methyl ester of the aliphatic amino acid tert-leucine 1 was investigated. This reaction path was compared with the electron transfer active amino acids methionine (N-acetyl methyl ester derivative 2a as well as the methyl ester derivative 2b) and the cysteine derivatives 3a and 3b (penicillamine derivative). Photofragmenation of the ester group dominated the photolysis of 1, whereas loss of methylmercaptane was observed for 2a and 3a. Vinylglycine derivatives 11 and 14 were formed to a minor extent. The gem-dimethylated compound 3b gave a solvent-characteristic product pattern with photoelimination products 18 and 19 in acetonitrile and loss of methylmercaptane to give 20 in methanol.

Photochemically catalyzed reaction of ochratoxin A with D- and L-cysteine.

The photolysis (>300 nm) of ochratoxin A (OTA, N-[[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl]carbonyl]-3-phenyl-L-alanine, 1) in the presence of excess (2 and 12 molar equiv) cysteine (CySH) has been investigated and found to yield sulfur adducts 5 and 6 that are characterized by liquid chromatography-mass spectrometry and 1H-NMR spectroscopy. The adduct 5 was ascribed to the Michael addition conjugate resulting from covalent attachment of CySH to the ochratoxin quinone (4) generated by photooxidation of OTA. This species was also formed by photolysis of a synthetic sample of the hydroquinone of OTA (ochratoxin hydroquinone, 3) in the presence of 12 equiv L-CySH. The conjugate 5 derived from photolysis of 3 with L-CySH was used for 1H-NMR analysis. The sulfur adduct 6 was the major species detected from covalent attachment of CySH to photoactivated OTA, and it resulted from direct displacement of the OTA Cl atom by CySH. The implications of the cysteinyl adducts to the in vivo toxicity of OTA are discussed, with particular emphasis given to conjugate 5, as products from the photooxidative pathway may be of relevance to the nephrotoxic properties of OTA.

Holmium: YAG lithotripsy: optimal power settings.

PURPOSE: We tested the hypothesis that holmium:YAG laser lithotripsy speed is best maximized by using low pulse energy at high pulse frequency. MATERIALS AND METHODS: To demonstrate that optical fiber damage increases with pulse energy and irradiation, the 365-microm optical fiber irradiated calcium hydrogen phosphate dihydrate (CHPD), calcium oxalate monohydrate (COM), cystine, magnesium ammonium phosphate hexahydrate (MAPH), and uric acid calculi at pulse energies of 0.5 to 2.0 J. Optical energy output was measured with an energy detector after 10 J to 200 J of total energy. To demonstrate that lithotripsy efficiency varies with power, fragmentation was measured at constant power settings at total energies of 200 J and 1 kJ with the 365-microm optical fiber. Fragmentation was measured for the 272-microm optical fiber at pulse energies of 0.5 J to 1.5 J at 10 Hz. To demonstrate that low pulse energy produces smaller fragments than high pulse energy, fragment size was characterized for COM and uric acid calculi after 0.25 kJ of irradiation using the 272-microm to 940-microm optical fibers at 0.5 J to 1.5 J. RESULTS: Damage to the 365-microm optical fiber was greatest for irradiation of CHPD, followed by MAPH, and COM (P<0.001). There was no significant optical fiber damage after cystine and uric acid lithotripsy. For the 365-microm optical fiber and CHPD, fragmentation after 200 J was greatest for pulse energies < or =1.0 J (P< 0.001). For other compositions, fragmentation was not statistically different among the power settings for constant irradiation. No significant difference was noted in fragmentation for any composition at different pulse energies (1.0 v. 2.0 J) for 1-kJ irradiation. However, for all compositions, the calculated lithotripsy speed was greatest at high power settings (P<0.001). For the 272-microm optical fiber, CHPD fragmentation was greatest for the 1.0-J pulse energy. The mean fragment size and relative quantity of fragments > or =2 mm both increased as pulse energy increased. CONCLUSIONS: Optical fiber degradation varies with stone composition, irradiation, and pulse energy. Holmium:YAG lithotripsy speed is maximized with higher power (either increased pulse energy or higher pulse frequency). Because low pulse energy may be safer and yields smaller fragments than high pulse energy, holmium:YAG lithotripsy speed is best increased by using pulse energies < or =1.0 J at a high repetition rate.

[New approach to the study of interaction of amino acid side groups with aryl azides]. / Novyi podkhod k izucheniiu vzaimodeistviia aminokislot po ikh bokovym radikalam s arilazidami.

A new approach to the study of the interaction of amino acid side chains with photoreactive aryl azides was proposed. This approach was based on the drawing together of the reacting groups by the attachment of the reacting compounds to complementary oligonucleotides. Cystamine, histamine, and 1,6-hexamethylenediamine mimicking the cystine, histidine, and lysine residues, respectively, were attached to the 3'-terminal phosphate of the oligonucleotide GGTATCp through a phosphamide bond and used as the targets for photomodification. Derivatives of the oligonucleotide pGATACCAA with the fragment N3C6H4NH- attached directly to its 5'-end by a phosphamide bond or through the spacer -(CH2)nNH- (where n is 2, 4, and 6) were used as photoreagents. Their derivatives containing the same spacer and the N3C6F4CO-NH(CH2)3NH- or 2-N3,5-NO2-C6H3CO-NH(CH2)3NH- residues were also used. The duplexes were photomodified by irradiation with 300-350 nm wavelength light. The maximal yields of the photo-cross-linking were from 22 to 68%. The reagents containing p-azidoaniline residue were found to be the most effective toward the targets. The maximum yields of the photomodification products modeling the side chains of cysteine and lysine were found to vary from 40 to 67% and to depend on the length and the structure of the spacers used. The duplex with the target bearing the imidazole residue (the histidine model) manifested a yield decreased to 25%. This fact was in a good agreement with the data of computer modeling that indicated an unfavorable mutual displacement of the imidazole residue and the photoreactive group.

Preparation of polymeric urease discs by an electron beam irradiation technique.

A preparation method of immobilized urease discs by an electron beam irradiation technique was developed, and the relationship between enzyme activity and preparation conditions was investigated. The immobilized urease disc was a thin circular film (200 microm, 5 mm phi) that is useful for biomedical applications. The activity of urease irradiated with 1 Mrad at room temperature was protected by the presence of cysteine. The activity of the immobilized urease discs was studied as a function of monomer concentration (80-90%) and the thicker disc gave a high activity. The durability of the immobilized urease discs gave a high activity. The durability of the immobilized urease discs was evaluated by repeated batch enzyme reactions, and a high activity yield (80-85%) was obtained.

Detection of free radicals in UV-irradiated lens by spin trapping ESR.

We have made an ESR study on the UV-photolysis of lens and identified the origins of free radicals involved in the initial photochemical process by spin trapping technique. Two spin adducts were detected on irradiation of canine lens in the presence of a spin trapping reagent (DMPO); a spin adduct of sulfur centered radical derived from glutathione and the protonated adduct of hydrated electron. A free radical mechanism of initial photochemical injury in UV-irradiated lens was discussed, comparing with a photolysis of tryptophan plus cysteine solution.

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186.

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.