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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Acceleration of deposition of Aß(1-40) peptide on ultrasonically formed Aß(1-42) nucleus studied by wireless quartz-crystal-microbalance biosensor.

High-frequency (~ 55 MHz) wireless quartz-crystal microbalance biosensor was used for studying heterogeneous deposition behavior of Aß(1-40) peptide on Aß(1-42) nuclei, which were grown under the stirring agitation and 200-kHz ultrasonication at pH 2.2, 4.6, and 7.4. The deposition reaction was monitored over 40 h, and the deposition rate was deduced. Among the agitation nuclei, the maximum deposition rate was observed on the nucleus grown at pH 4.6. However, ultrasonication nucleus grown at pH 7.4 produced much larger deposition rate, despite the same ß-sheet concentration. This result indicates that local structural modulation is caused in the nucleus by ultrasonication, which adsorbs the Aß peptide more actively than other nuclei. The resultant deposits clearly show oligomeric structure.

Photocleavage of avidin by a new pyrenyl probe.

In this study, a new small-molecule-based reagent was designed to recognize and bind to specific site in protein. A new pyrenyl probe, d-biotinyl-1(1-pyrene)methylamide (Py-biotin) was designed and synthesized by coupling of d-biotin to 1(1-pyrene)methylamine hydrochloride. Binding studies and site-specific photocleavage of avidin by Py-biotin were demonstrated. Binding of Py-biotin to avidin was studied using absorbance and fluorescence spectroscopic techniques. Red shifts of the absorption peak positions of the pyrenyl chromophore followed by hyperchromism were observed upon binding to avidin. The photocleavage of avidin was achieved when a mixture of the protein, Py-biotin, and an electron acceptor, cobalt(III) hexammine trichloride (CoHA), was irradiated at 342nm. No reaction occurred in the absence of the probe, CoHA, or light. N-terminal sequencing of the peptide fragments indicated a cleavage site of avidin between Thr 77 and Val 78. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

Photoactivable peptides for identifying enzyme-substrate and protein-protein interactions.

Photoactivated cross-linking of peptides to proteins is a useful strategy for identifying enzyme-substrate and protein-protein interactions in cell lysates as demonstrated by studies on the human hypoxia inducible factor system.

Low energy laser light (632.8 nm) suppresses amyloid-ß peptide-induced oxidative and inflammatory responses in astrocytes.

Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid ß-peptides (Aß) in mediating these processes. In astrocytes, oligomeric Aß induces the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complexes resulting in its activation to produce anionic superoxide. Aß also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aß. Primary rat astrocytes were exposed to Helium-Neon laser (λ=632.8 nm), followed by the treatment with oligomeric Aß. Primary rat astrocytes were used to measure Aß-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47(phox) subunit and the membrane gp91(phox) subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A(2) cPLA(2) and expressions of pro-inflammatory factors including interleukin-1ß (IL-1ß) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aß-induced superoxide production, colocalization between NADPH oxidase gp91(phox) and p47(phox) subunits, phosphorylation of cPLA(2,) and the expressions of IL-1ß and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD.

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.

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.