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.

Optical Control of Antibody Activity by Using Photocleavable Bivalent Peptide-DNA Locks.

Antibody-based molecular recognition plays a central role in today's life sciences, ranging from immunoassays to molecular imaging and antibody-based therapeutics. Control over antibody activity by using external triggers such as light could further increase the specificity of antibody-based targeting. Here we present bivalent peptide-DNA ligands containing photocleavable linkers as a noncovalent approach by which to allow photoactivation of antibody activity. Light-triggered cleavage of the 3-amino-3-(2-nitrophenyl)propionic acid peptide linker converted the high-affinity bivalent peptide-DNA lock into weakly binding monovalent ligands, effectively restoring antibody targeting of cell-surface receptors. In this work, a proof of principle was provided with an anti-hemagglutinin antibody, but the molecular design of the lock is generic and applicable to any monoclonal antibody for which an epitope or mimotope of sufficient affinity is available.

Sequence-specific formation of d-amino acids in a monoclonal antibody during light exposure.

The photoirradiation of a monoclonal antibody 1 (mAb1) at λ = 254 nm and λmax = 305 nm resulted in the sequence-specific generation of d-Val, d-Tyr, and potentially d-Ala and d-Arg, in the heavy chain sequence [95-101] YCARVVY. d-Amino acid formation is most likely the product of reversible intermediary carbon-centered radical formation at the (α)C-positions of the respective amino acids ((α)C(•) radicals) through the action of Cys thiyl radicals (CysS(•)). The latter can be generated photochemically either through direct homolysis of cystine or through photoinduced electron transfer from Trp and/or Tyr residues. The potential of mAb1 sequences to undergo epimerization was first evaluated through covalent H/D exchange during photoirradiation in D2O, and proteolytic peptides exhibiting deuterium incorporation were monitored by HPLC-MS/MS analysis. Subsequently, mAb1 was photoirradiated in H2O, and peptides, for which deuterium incorporation in D2O had been documented, were purified by HPLC and subjected to hydrolysis and amino acid analysis. Importantly, not all peptide sequences which incorporated deuterium during photoirradiation in D2O also exhibited photoinduced d-amino acid formation. For example, the heavy chain sequence [12-18] VQPGGSL showed significant deuterium incorporation during photoirradiation in D2O, but no photoinduced formation of d-amino acids was detected. Instead this sequence contained ca. 22% d-Val in both a photoirradiated and a control sample. This observation could indicate that d-Val may have been generated either during production and/or storage or during sample preparation. While sample preparation did not lead to the formation of d-Val or other d-amino acids in the control sample for the heavy chain sequence [95-101] YCARVVY, we may have to consider that during hydrolysis N-terminal residues (such as in VQPGGSL) may be more prone to epimerization. We conclude that the photoinduced, radical-dependent formation of d-amino acids requires not only the intermediary formation of a (α)C(•) radical but also sufficient flexibility of the protein domain to allow both pro-chiral faces of the (α)C(•) radical to accept a hydrogen atom.

In vitro electrochemical detection of the degradation of amyloid-ß oligomers.

The clearance of overloaded amyloid ß (Aß) oligomers is thought to be an attractive and potential strategy for the therapy of Alzheimer's disease (AD). A variety of strategies have already been utilized to study Aß degradation in vitro. Here, the electrochemical detection based on direct electrooxidation of specific Tyr residues within Aß peptide has been developed as a simple and robust approach for monitoring the oligomers' degradation. C60 was employed for photodegrading Aß oligomers due to the generated ROS under light irradiation. The oxidation current of Tyr residues by square wave voltammetry (SWV) increased upon the Aß degradation, confirming that the structure variation of Aß peptide indeed influenced the exposure of those redox species to the electrode surface and final signal output. Chronoamperometric assay also found the electrooxidation of Tyr undergone an irreversible process. Additionally, the direct electrochemistry was capable of detecting the aggregation with rapid test and better sensitivity in compared with dynamic light scattering (DLS), atomic force microscopy (AFM) and thioflavin T (ThT) based fluorescence assay. Thus, this work indicated the potential application of direct electrochemistry in the in vitro measurement of Aß degradation and clearance, providing new insights and a complementary means into the AD theranostics.

Probing d- and l-Adrenaline Binding to ß2-Adrenoreceptor Peptide Motifs by Gas-Phase Photodissociation Cross-Linking and Ion Mobility Mass Spectrometry.

Diazirine-tagged d- and l-adrenaline derivatives formed abundant noncovalent gas-phase ion complexes with peptides N-Ac-SSIVSFY-NH2 (peptide S) and N-Ac-VYILLNWIGY-NH2 (peptide V) upon electrospray ionization. These peptide sequences represent the binding motifs in the ß2-adrenoreceptor. The structures of the gas-phase complexes were investigated by selective laser photodissociation of the diazirine chromophore at 354 nm, which resulted in a loss of N2 and formation of a transient carbene intermediate in the adrenaline ligand without causing its expulsion. The photolyzed complexes were analyzed by collision-induced dissociation (CID-MS3 and CID-MS4) in an attempt to detect cross-links and establish the binding sites. However, no cross-linking was detected in the complexes regardless of the peptide and d- or l-configuration in adrenaline. Cyclic ion mobility measurements were used to obtain collision cross sections (CCS) in N2 for the peptide S complexes. These showed identical values, 334 ± 0.9 Å2, for complexes of the l- and d-adrenaline derivatives, respectively. Identical CCS were also obtained for peptide S complexes with natural l- and d-adrenaline, 317 ± 1.2 Å2, respectively. Born-Oppenheimer molecular dynamics (BOMD) in combination with full geometry optimization by density functional theory calculations provided structures for the complexes that were used to calculate theoretical CCS with the ion trajectory method. A close match (337 Å2) was found for a single low Gibbs energy structure that displayed a binding pocket with Ser 2 and Ser 5 residues forming hydrogen bonds to the adrenaline catechol hydroxyls. Analysis of the BOMD trajectories revealed a small number of contacts between the incipient carbene carbon atom in the ligand and X-H bonds in the peptide, which was consistent with the lack of cross-linking. Temperature dependence of the internal dynamics of peptide S-adrenaline complexes as well as the specifics of the adrenaline carbene reactions are discussed. In particular, peptide amide hydrogen transfer to the carbene carbon atom was calculated to require crossing a potential energy barrier, which may hamper cross-linking in competition with carbene internal rearrangements.

Blue light induces global and localized conformational changes in the kinase domain of full-length phototropin.

The blue-light photoreceptor phototropin plays a crucial role in optimizing photosynthesis in plants. In the two light-, oxygen-, or voltage-sensitive (LOV) domains of phototropin, the light stimulus is absorbed by the flavin chromophores. The signal is assumed to be transferred via dissociation and unfolding of a conserved J alpha helix element to the serine/threonine kinase domain. We investigated full-length phototropin from the green alga Chlamydomonas reinhardtii by Fourier transform infrared spectroscopy to shed light on the signal transfer within the protein and on the structural response of the kinase. Light-induced structural changes were assigned by comparing signals of the full-length protein with those of the truncated LOV1-LOV2-J alpha and LOV1-LOV2 and with those of deletion mutants. A loss of helicity originating from the J alpha linker helix was observed in LOV1-LOV2-J alpha in agreement with previous studies of LOV2-J alpha. Full-length phototropin showed reversible global conformational changes via several turn elements. These changes were suppressed in a deletion mutant lacking the J alpha linker and are attributed to the kinase domain. The loss of turn structure is interpreted as a light-induced opening of the kinase tertiary structure upon release of the LOV2 domain. Concomitant protonation changes of Asp or Glu residues in the kinase domain were not observed. A light-induced loss in helicity was observed only in the presence of a phototropin-characteristic 54-amino acid extension of the kinase activation loop, which is predicted to be located apart from the catalytic cleft. This response of the extension might play a significant role in the phototropin signaling process.

Modeling, preparation, and characterization of a dipole moment switch driven by Z/E photoisomerization.

We report the results of a multidisciplinary research effort where the methods of computational photochemistry and retrosynthetic analysis/synthesis have contributed to the preparation of a novel N-alkylated indanylidene-pyrroline Schiff base featuring an exocyclic double bond and a permanent zwitterionic head. We show that, due to its large dipole moment and efficient photoisomerization, such a system may constitute the prototype of a novel generation of electrostatic switches achieving a reversible light-induced dipole moment change on the order of 30 D. The modeling of a peptide fragment incorporating the zwitterionic head into a conformationally rigid side chain shows that the switch can effectively modulate the fluorescence of a tryptophan probe.

Aggregation of amyloid Abeta((1-40)) peptide in perdeuterated 2,2,2-trifluoroethanol caused by ultrasound sonication.

Ultrasound sonication of protein and peptide solutions is routinely used in biochemical, biophysical, pharmaceutical and medical sciences to facilitate and accelerate dissolution of macromolecules in both aqueous and organic solvents. However, the impact of ultrasound waves on folding/unfolding of treated proteins, in particular, on aggregation kinetics of amyloidogenic peptides and proteins is not understood. In this work, effects of ultrasound sonication on the misfolding and aggregation behavior of the Alzheimer's Abeta((1-40))-peptide is studied by pulsed-field gradient (PFG) spin-echo diffusion NMR and UV circular dichroism (CD) spectroscopy. Upon simple dissolution of Abeta((1-40)) in perdeuterated trifluoroethanol, CF(3)-CD(2)-OD (TFE-d(3)), the peptide is present in the solution as a stable monomer adopting alpha-helical secondary structural motifs. The self-diffusion coefficient of Abeta((1-40)) monomers in TFE-d(3) was measured as 1.35 x 10(-10) m(2) s(-1), reflecting its monomeric character. However, upon ultrasonic sonication for less than 5 min, considerable populations of Abeta molecules (ca 40%) form large aggregates as reflected in diffusion coefficients smaller than 4.0 x 10(-13) m(2) s(-1). Sonication for longer times (up to 40 min in total) effectively reduces the fraction of these aggregates in (1)H PFG NMR spectra to ca 25%. Additionally, absorption below 230 nm increased significantly upon sonication treatment, an observation, which also clearly confirms the ongoing aggregation process of Abeta((1-40)) in TFE-d(3). Surprisingly, upon ultrasound sonication only small changes in the peptide secondary structure were detected by CD: the peptide molecules mainly adopt alpha-helical motifs in both monomers and aggregates formed upon sonication.

Ultraviolet Photodissociation of Tryptic Peptide Backbones at 213 nm.

We analyzed the backbone fragmentation behavior of tryptic peptides of a four-protein mixture and of E. coli lysate subjected to ultraviolet photodissociation (UVPD) at 213 nm on a commercially available UVPD-equipped tribrid mass spectrometer. We obtained 15 178 unique high-confidence peptide UVPD spectrum matches by recording a reference beam-type collision-induced dissociation (HCD) spectrum of each precursor, ensuring that our investigation includes a broad selection of peptides, including those that fragmented poorly by UVPD. Type a, b, and y ions were most prominent in UVPD spectra, and median sequence coverage ranged from 5.8% (at 5 ms laser excitation time) to 45.0% (at 100 ms). Overall, the sequence fragment intensity remained relatively low (median: 0.4% (5 ms) to 16.8% (100 ms) of total intensity), and the remaining precursor intensity, high. The sequence coverage and sequence fragment intensity ratio correlated with the precursor charge density, suggesting that UVPD at 213 nm may suffer from newly formed fragments sticking together due to noncovalent interactions. The UVPD fragmentation efficiency therefore might benefit from supplemental activation, as was shown for ETD. Aromatic amino acids, most prominently tryptophan, facilitated UVPD. This points to aromatic tags as possible enhancers of UVPD. Data are available via ProteomeXchange with identifier PXD018176 and on spectrumviewer.org/db/UVPD-213nm-trypPep.

Solution structure of Psb27 from cyanobacterial photosystem II.

Psb27 is a highly conserved component of photosystem II. The three-dimensional structure has a well-defined helical core, composed of four helices arranged in a right-handed up-down-up-down fold, with a less ordered region of the structure located at the N-terminus. The position of conserved residues on the surface suggests conserved functional roles for distinct interconnected features encompassing a P-phi-P loop, a polar patch spanning helices alpha3 and alpha4, and the N-terminal sequence.

The N-terminal region of photocleavable peptides that bind HLA-DR1 determines the kinetics of fragment release.

Major Histocompatibility Complex class II (MHC-II) molecules bind peptides and present them to receptors on CD4+ T cells as part of the immune system's surveillance of pathogens and malignancy. In the absence of peptide, MHC-II equilibrates between peptide-receptive and peptide-averse conformations. The conversion between these forms has been postulated to be important in regulating cellular antigen presentation but has been difficult to study. In order to generate the MHC-II molecule HLA-DR1 in the peptide-receptive form, we designed and tested a series of photocleavable peptides that included the UV-sensitive 3-amino-3-(2-nitrophenyl)-propionate amino acid analog. They were intended to bind tightly to the HLA-DR1 MHC molecule, but to generate low-affinity fragments after UV exposure that would be released to yield HLA-DR1 in the peptide-receptive conformation. We were able to identify photocleavable peptides that bound tightly to HLA-DR1 and generated the peptide-receptive conformation after UV exposure. However, slow release of photocleaved peptide fragments from the binding site limited the rate of binding of an incoming labeled peptide and complicated kinetic measurements of the individual steps of the overall peptide binding reaction. Modification of the N-terminal region of the photocleavable peptide to reduce MHC-II pocket or H-bonding interactions allowed for generation of the peptide receptive form immediately after UV exposure with peptide fragments neither retained within the site nor interfering with binding of an incoming peptide. However this was achieved only at the expense of a substantial reduction in overall peptide binding affinity, and these peptides had such weak interaction with HLA-DR1 that they were easily exchanged by incoming peptide without UV exposure. These results show that photocleavable peptides can be used to generate peptide-receptive HLA-DR1 and to facilitate peptide exchange in generation of specific peptide-MHC-II complexes, but that usage of these peptides for kinetic studies can be constrained by slow fragment release.

Modular design of optically controlled protein affinity reagents.

Photopharmaceuticals can, in principle, be created by linking photoswitchable moieties to bioactive molecules. However, a general strategy for converting a therapeutic agent into its photoswitchable version is not currently available. Herein we propose a generalizable, modular approach for obtaining light controllable bioactive agents by modifying the scaffold of a protein affinity reagent using an azobenzene photoswitch.

Protective effects of mycosporine-like amino acids of Synechocystis sp. PCC 6803 and their partial characterization.

In this work, mycosporine-like amino acids (MAAs) of Synechocystis sp. PCC 6803 were characterized and were investigated on UV induction and protective ability. High performance liquid chromatographic (HPLC) studies revealed three major compounds in the MAAs. By UV absorption and mass spectra analysis, one of the compounds was tentatively identified as mycosporine-tau (M-tau). One novel compound similar to usujirene was tentatively named as dehydroxylusujirene, and the other novel compound was named as M-343 according to its absorption maximum. In vivo experiments indicated that M-tau was induced by both UV-A and UV-B, while dehydroxylusujirene and M-343 were only induced by UV-A, suggesting that different chromophores were involved in MAAs synthesis in Synechocystis sp. PCC 6803. It was also indicated that M-343 could be photochemically synthesized from some precursors. Under both UV and oxidation stresses, M-343 was more stable than dehydroxylusujirene and M-tau. Considering the reaction with H2O2, M-tau and dehydroxylusujirene might be potential antioxidants in reaction with physiological reactive oxygen species in vivo. In protection experiments, the MAAs exhibited efficient protective ability towards UV-B and H2O2 stresses, with maximal protection rates of 30% and 21.5%, respectively. These results indicate that the MAAs in Synechocystis sp. PCC 6803 act as both UV-screen and antioxidant.

Light-driven porphyrin modulating fibrillation of hIAPP(20-29) peptide.

The human Islet amyloid polypeptide (20-29) (hIAPP20-29) is considered to be the core fibrillating fragment of hIAPP, which is associated with the pathogenesis of Type-II diabetes mellitus. A current challenge is the discovery of an efficient way to modulate amyloid aggregation and inhibit the toxicity of its aggregates. In this work, photoexcited porphyrins are successfully used to inhibit the fibrillation of hIAPP20-29. Insights on the inhibitory mechanism are explored by the analysis of the secondary structure, the morphology and the mechanical properties of amyloid aggregates. In addition, photoexcited porphyrins displayed a retained inhibitory effect on hIAPP20-29 aggregation without irradiation. These findings may establish a new avenue to inhibit the aggregation of amyloid peptide hIAPP and enrich the current selection of modulators.

Antioxidant activity measurement and potential antioxidant peptides exploration from hydrolysates of novel continuous microwave-assisted enzymolysis of the Scomberomorus niphonius protein.

A novel continuous microwave-assisted enzymatic digestion (cMAED) method is proposed for the digestion of protein from Scomberomorus niphonius to obtain potential antioxidant peptides. In this study, bromelain was found to have a high capacity for the digestion of the Scomberomorus niphonius protein. The following cMAED conditions were investigated: protease species, microwave power, temperature, bromelain content, acidity of the substrate solution, and incubation time. At 400W, 40°C, 1500U·g-1 bromelain, 20% substrate concentration, pH 6.0 and 5min incubation, the degree of hydrolysis and total antioxidant activity of the hydrolysates were 15.86% and 131.49µg·mL-1, respectively. The peptide analyses showed that eight of the potential antioxidant peptide sequences, which ranged from 502.32 to 1080.55Da with 4-10 amino acid residues, had features typical of well-known antioxidant proteins. Thus, the new cMAED method can be useful to obtain potential antioxidant peptides from protein sources, such as Scomberomorus niphonius.

Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation.

The yellow fluorescent protein (YFP) is frequently used in a protein complementation assay called bimolecular fluorescence complementation (BiFC), and is employed to visualize protein-protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein-protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein-protein interactions. Some spectral characteristics of "multicolor" BiFC (mcBiFC) complexes have been reported before; however, no in-depth analysis has been performed yet. Therefore, little is known about the photophysical characteristics of these mcBiFC complexes because a proper characterization essentially relies on in vitro data. This is particularly difficult for fragments of autofluorescent proteins (AFPs) because they show a very strong tendency to form supramolecular aggregates which precipitate ex vivo. In this study, this intrinsic difficulty is overcome by directly fusing the coding DNA of different AFP fragments. Translation of the genetic sequence in Escherichia coli leads to fully functional, highly soluble fluorescent proteins with distinct properties. On the basis of their construction, they are designated chimeric AFPs, or BiFC chimeras, here. Comparison of their spectral characteristics with experimental in vivo BiFC data confirmed the utility of the chimeric proteins as a BiFC model system. In this study, nine different chimeras were thoroughly analyzed at both the ensemble and the single-molecular level. The data indicates that mutations believed to be photophysically silent significantly alter the properties of AFPs.

Azobenzene photocontrol of peptides and proteins.

The last few years have witnessed significant advances in the use of light as a stimulus to control biomolecular interactions. Great efforts have been devoted to the development of genetically encoded optobiological and small photochromic switches. Newly discovered small molecules now allow researchers to build molecular systems that are sensitive to a wider range of wavelengths of light than ever before with improved switching fidelities and increased lifetimes of the photoactivated states. Because these molecules are relatively small and adopt predictable conformations they are well suited as tools to interrogate cellular function in a spatially and temporally contolled fashion and for applications in photopharmacology.

Increased p53 phosphorylation after microtubule disruption is mediated in a microtubule inhibitor- and cell-specific manner.

p53 is present at low levels in unstressed cells. Numerous cellular insults, including DNA damage and microtubule disruption, elevate p53 protein levels. Phosphorylation of p53 is proposed to be important for p53 stabilization and activation after genotoxic stress; however, p53 phosphorylation after microtubule disruption has not been analysed. The goal of the current study was to determine if p53 phosphorylation increases after microtubule disruption, and if so, to identify specific p53 residues necessary for microtubule inhibitor-induced phosphorylation. Two dimensional gel analyses demonstrated that the number of p53 phospho-forms in cells increased after treatment with microtubule inhibitors (MTIs) and that the pattern of p53 phosphorylation was distinct from that observed after DNA damage. p53 phosphorylation also varied in a MTI-dependent manner, as Taxol and Vincristine induced more p53 phospho-forms than nocodazole. Further, MTI treatment increased phosphorylation of p53 on serine-15 in epithelial tumor cells. In contrast, serine-15 phosphorylation of p53 did not increase in MTI-treated primary cultures of human fibroblasts. Analysis of ectopically expressed p53 phospho-mutant proteins from Taxol- and nocodazole-treated cells indicated that multiple p53 amino terminal residues, including serine-15 and threonine-18, were required for Taxol-mediated phosphorylation of p53. Taken together, the results of this study demonstrate that distinct p53 phospho-forms are induced by MTI treatment as compared to DNA damage and that p53 phosphorylation is mediated in a MTI- and cell-specific manner. Oncogene (2001) 20, 113 - 124.

Photo-induced inhibition of Alzheimer's ß-amyloid aggregation in vitro by rose bengal.

The abnormal aggregation of ß-amyloid (Aß) peptides in the brain is a major pathological hallmark of Alzheimer's disease (AD). The suppression (or alteration) of Aß aggregation is considered to be an attractive therapeutic intervention for treating AD. We report on visible light-induced inhibition of Aß aggregation by xanthene dyes, which are widely used as biomolecule tracers and imaging markers for live cells. Among many xanthene dyes, rose bengal (RB) under green LED illumination exhibited a much stronger inhibition effect upon photo-excitation on Aß aggregation than RB under dark conditions. We found that RB possesses high binding affinity to Aß; it exhibits a remarkable red shift and a strong enhancement of fluorescence emission in the presence of Aß. Photo-excited RB interfered with an early step in the pathway of Aß self-assembly and suppressed the conformational transition of Aß monomers into ß-sheet-rich structures. Photo-excited RB is not only effective in the inhibition of Aß aggregation, but also in the reduction of Aß-induced cytotoxicity.

Cleavage and phosphorylation of XRCC4 protein induced by X-irradiation.

We report the p35 and p60 forms of XRCC4 protein, appearing in human leukemia MOLT-4 or U937 cells following X-irradiation or hyperthermia. p35 appeared in conjunction with the cleavage of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the fragmentation of internucleosomal DNA, and was suppressed by Ac-DEVD-CHO. p35 was also produced in vitro by treating MOLT-4 cell lysate with recombinant caspases, suggesting that p35 was a caspase-cleaved fragment of XRCC4 in apoptotic cell death. p60 was sensitive to treatment with phosphatase or wortmannin and was undetectable in M059J cells deficient in DNA-PKcs. However, p60 was found in ataxia-telangiectasia cells after irradiation. These results indicated p60 as a phosphorylated form of XRCC4, requiring DNA-PKcs but not ataxia-telangiectasia mutated (ATM).