Enzymatically modified peptide surfaces: towards general electrochemical sensor platform for protein kinase catalyzed phosphorylations

We hereby present an electrochemical approach for monitoring the three protein kinases sarcoma-related kinase (Src), extracellular signal-regulated kinase 1 (Erk1), and cyclin A-dependent kinase 2 (CDK2/cyclin A). The electrochemical sensor is based on the ability of kinases to transfer a redox-labeled phosphoryl group to surface-bound peptides that are highly specific substrates for the particular protein kinase (EGIYDVP, EPLTPSG, and HHASPRK, respectively). The detection method relies on the use of 5'-γ-ferrocenoyl-ATP (Fc-ATP) as a co-substrate for peptide phosphorylation. The peptides themselves are attached to a Au substrate, which acts as the working electrode. In this process a Fc-phosphoryl group is transferred to the peptide and the presence of the redox active Fc group is detected electrochemically. All peptide films were fully characterized by cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). Particular attention was given to the electron transfer rates, k(ET), in peptide films after Fc-phosphorylation which were found to be on the order of seconds. The slow ET kinetics is presumably a result of the negative charge on the phosphoryl group. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) experiments based on the peptide modified Au surfaces reveal significant ferrocene and phosphate group content introduced using the kinase-catalyzed phosphorylation reaction.     

Synthesis and spectral investigations of a new dyad with spiropyran and fluorescein units: toward information processing at the single molecular level

A new dyad 1 with two spiropyran units as the photochromic acceptors and one fluorescein unit as the fluorescent donor was synthesized and characterized. External inputs (ultraviolet light, visible light, and proton) induce the reversible changes of the structure and, concomitantly, the absorption spectrum of dyad 1 due to the presence of two spiropyran units. Only the absorption spectrum of the ME form of the spiropyran units in dyad 1 has large spectral overlap with the fluorescence spectrum of the fluorescein unit. Thus, the fluorescence intensity of dyad 1 is modulated by reversible conversion among the three states of the photochromic spiropyran units and the fluorescence resonance energy transfer (FRET) between the ME form and the fluorescein unit. Based on the fact that dyad 1 could "read out" three external input signals (ultraviolet light, visible ligh,t and proton) and "write" a compatible specific output signal (fluorescence intensity), dyad 1 described here can be considered to perform an integrated circuit function with one OR and one AND interconnected logic gates. The present results demonstrate an efficient strategy for elaborating and transmitting information at the single molecular level.     

Determination of protein phosphorylation by extracellular signal-regulated kinase using capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Extracellular signal-regulated kinase (ERK) is a key regulatory enzyme mediating cell responses to mitogenic stimulation and is one of the key components in linking growth factor receptor activation to serine/threonine protein phosphorylation processes. Phosphorylation reaction by ERK plays an important role in many signal transduction pathways. ERK phosphorylates numerous substrates such as MBP, microtubule-associated protein 2 (MAP2) and nuclear protein. In particular, MBP is a substrate commonly employed for the detection of ERK activity and contains the consensus primary sequence PRT97P. In this paper, we compared the degree of the phosphorylation reaction of MBP substrate peptides by ERK with the three different MBP substrate peptides, MBP1(KNIVTPRTPPPSQGK), MBP2(VPRTPGGRR) and MBP3(APRTPGGRR) in order to select an efficient substrate peptide for phosphorylation reaction by ERK. The results showed that the MBP3 peptide is the most efficient substrate for phosphorylation reaction by ERK. Using MBP3 peptide, the phosphorylation reaction of MBP by ERK was monitored with both matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis (CE). Our results demonstrate the feasibility of the CE method, the method being a simple and reliable technique in determining and characterizing various kinds of enzyme reaction especially including kinase enzymes.     

New Generation of Tunable Bioactive Shape Memory Mats Integrated with Genetically Engineered Proteins

Aligned poly(l ‐lactide)/poly(methyl methacrylate) binary blend fibers and mats loaded with a chimeric green fluorescence protein having a bioactive peptide with hydroxyapatite binding and mineralization property are prepared by pressurized gyration. The effect of processing parameters on the product morphologies, and the shape memory properties of these samples are investigated. Integration of hydroxyapatite nanoparticles into the fiber assembly is self‐directed using the hydroxyapatite‐binding property of the peptide genetically engineered to green fluorescence protein. Fluorescence microscopy analysis corroborated with Fourier transform infrared spectroscopy (FTIR) data confirms the integration of the chimeric protein with the fibers. An enzyme based remineralization assay is conducted to study the effects of peptide‐mediated mineralization within the fiber mats. Raman and FTIR spectral changes observed following the peptide‐mediated mineralization provides an initial step toward a soft‐hard material transition. These results show that programmable shape memory properties can be obtained by incorporating genetically engineered bioactive peptide domains into polymer fibers.

     

Mass spectrometric approach for the analysis of food proteins

In the study of food proteins, the need for accurate protein structural analysis has been acknowledged because of the fact that nucleotide sequencing alone is of limited analytical value if not combined with relevant information regarding the specific protein expressed and the occurrence of phosphorylation, glycosylation and disulphide bridges, and with the modification induced by the technological treatment. Mass spectrometry, whether used alone or to complement the traditional molecular-based techniques has become fundamental to the structural analysis of proteins. It is, moreover, virtually irreplaceable in determining post-translational modifications as conventional methods cannot deliver reliable data. What lies at the root of this methodological breakthrough is the combination of high-resolution separation techniques such as two-dimensional electrophoresis or capillary reverse- phase high-performance liquid chromatography with mass spectrometric analysis, what is termed "proteomic" analysis. Thus, it appears appropriate to state that the new mass spectrometric techniques have been established as a valuable and efficient tool for protein and peptide analysis in complex mixtures, like those from food matrices, enabling us therefore to provide accurate information on molecular weight and also to put forth a structural assessment at a low-picomole level of material. Thus, a series of alternative approaches have been developed based on advanced mass spectrometric analysis in conjunction with classic protein chemistry in order to provide an in-depth view of food protein structure. This review outlines several of these novel methodologies as they apply to structural characterization of food products.     

Memory effects based on intermolecular photoinduced proton transfer

We have identified a strategy to communicate a chemical signal between two independent molecular components. One of them is a photoactive merocyanine that switches to a spiropyran, releasing a proton, when stimulated with visible light. The other is a 4,4'-pyridylpyridinium monocation that captures the released proton, producing an electroactive 4,4'-bipyridinium dication. Under the irradiation conditions employed, the photoinduced transformation requires ca. 15 min to reach a photostationary state. In the dark, the ensemble of communicating molecules reequilibrates to the original state in ca. 5 days. These processes can be monitored following the photoinduced enhancement and thermal decay, respectively, of the current for the monolectronic reduction of the 4,4'-bipyridinium dication. The pronounced difference in time scale for the current enhancement and decay steps can be exploited to implement a memory element with a bit retention time of 11 h. A bit of information can be written optically in the chemical system and it can be read electrically and nondestructively. The memory can be reset, extending its permanence in the dark beyond the bit retention time. A binary logic analysis of the signal transduction operated by the communicating molecules reveals the characteristic behavior of sequential logic operators, which are the basic components of digital memories.     

A Reversible Multi‐Stimuli‐Responsive Fluorescence Probe and the Design for Combinational Logic Gate Operations

Here, a novel multi‐stimuli‐responsive fluorescence probe is developed by incorporating spiropyran group into the coumarin‐substituted polydiacetylene (PDA) vesicles. The fluorescence of PDA can be turned on upon heating, and can be quenched upon exposure to UV light irradiation or pH stimuli owing to the fluorescene resonance energy transfer (FRET) between the red‐phase PDA and the open merocyanine (MC) form of spiropyran. Moreover, we have designed and experimentally realized a set of logic gate operations for the first time based on the fluorescence modulation of the designed system upon thermal, photo, and pH stimuli. This novel type of resettable logic gates augur well for practical applications in information storage, optical recording, and sensing in complicated microenvironments.

     

Photoresponsive n-channel organic field-effect transistors based on a tri-component active layer

Photoresponsive OFETs were fabricated based on a tri-component active layer (NDI2OD-DTYM2, spiropyran and polystyrene). The results demonstrated that these OFETs displayed photoresponsive feature to alternate UV and vis light due to the photoisomerization of spiropyran between the closed-ring state and ionic open-ring state.     

Structure of a new photochromic indoline spiropyran

Conclusions An x-ray diffraction structural study was carried out for the photochromic spirpyran 1,3,3-trimethyl-5-nitrospiro(indoline-2,2-[2H-1]-benzopyran). The C_spiro-O bond in the spiropyran is longer than the normal value but shorter than in the isomeric spiropyran with the nitro group in the benzopyran fragment, which is characterized by a higher quantum yield.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 1, pp. 191–194, January, 1985.     

Coherent Activation of DNA Tweezers: A “SET–RESET” Logic System

Open sesame : Aptamer–substrate complexes activate the coherent operation of two tweezers that act as a “SET–RESET” logic system. Each tweezer cycles between a fluorescent open state and a closed quenched state (Q=quencher, F=fluorophore) when triggered by adenosine monophosphate (AMP) and adenosine deaminase (AD).

     

Peptide Logic Circuits Based on Chemoenzymatic Ligation for Programmable Cell Apoptosis

A novel and versatile peptide‐based bio‐logic system capable of regulating cell function is developed using sortase A (SrtA), a peptide ligation enzyme, as a generic processor. By modular peptide design, we demonstrate that mammalian cells apoptosis can be programmed by peptide‐based logic operations, including binary and combination gates (AND, INHIBIT, OR, and AND‐INHIBIT), and a complex sequential logic circuit (multi‐input keypad lock). Moreover, a proof‐of‐concept peptide regulatory circuit was developed to analyze the expression profile of cell‐secreted protein biomarkers and trigger cancer‐cell‐specific apoptosis.     

How does the cAMP-dependent protein kinase catalyze the phosphorylation reaction: an ab initio QM/MM study

We have carried out density functional theory QM/MM calculations on the catalytic subunit of cAMP-dependent protein kinase (PKA). The QM/MM calculations indicate that the phosphorylation reaction catalyzed by PKA is mainly dissociative, and Asp166 serves as the catalytic base to accept the proton delivered by the substrate peptide. Among the key interactions in the active site, the Mg(2+) ions, glycine rich loop, and Lys72 are found to stabilize the transition state through electrostatic interactions. On the other hand, Lys168, Asn171, Asp184, and the conserved waters bound to Mg(2+) ions do not directly contribute to lower the energy barrier of the phosphorylation reaction, and possible roles for these residues are proposed. The QM/MM calculations with different QM/MM partition schemes or different initial structures yield consistent results. In addition, we have carried out 12 ns molecular dynamics simulations on both wild type and K168A mutated PKA, respectively, to demonstrate that the catalytic role of Lys168 is to keep ATP and substrate peptide in the near-attack reactive conformation.     

Ultrafast UV-mid-IR investigation of the ring opening reaction of a photochromic spiropyran

We present a femtosecond UV-mid-IR pump-probe study of the photochemical ring-opening reaction of the spiropyran 1',3',3',-trimethylspiro-[-2H-1-benzopyran-2,2'-indoline] (also known as BIPS) in tetrachloroethene, using 70 fs UV excitation pulses and probing with 100 fs mid-IR pulses. The time evolution of the transient IR absorption spectrum was monitored over the first 100 ps after UV excitation. We conclude that the merocyanine product is formed with a 28 ps time constant, contrasting with a 0.9 ps time constant obtained in previous investigations where the rise of absorption bands at visible wavelengths were associated with product formation. We deduce from the observed strong recovery of the spiropyran IR absorption bleaches that, in tetrachloroethene, the main decay channel for the S(1) excited state of the spiropyran BIPS, is internal conversion to the spiropyran S(0) state with a quantum yield of > or = 0.9. This puts an upper limit of 0.1 to the quantum yield of the photochemical ring-opening reaction.     

Spatially resolved Raman spectroelectrochemistry of solid-state polythiophene/viologen memory devices

A three terminal molecular memory device was monitored with in situ Raman spectroscopy during bias-induced switching between two metastable states having different conductivity. The device structure is similar to that of a polythiophene field effect transistor, but ethylviologen perchlorate was added to provide a redox counter-reaction to accompany polythiophene redox reactions. The conductivity of the polythiophene layer was reversibly switched between high and low conductance states with a "write/erase" (W/E) bias, while a separate readout circuit monitored the polymer conductance. Raman spectroscopy revealed reversible polythiophene oxidation to its polaron form accompanied by a one-electron viologen reduction. "Write", "read", and "erase" operations were repeatable, with only minor degradation of response after 200 W/E cycles. The devices exhibited switching immediately after fabrication and did not require an "electroforming" step required in many types of memory devices. Spatially resolved Raman spectroscopy revealed polaron formation throughout the polymer layer, even away from the electrodes in the channel and drain regions, indicating that thiophene oxidation "propagates" by growth of the conducting polaron form away from the source electrode. The results definitively demonstrate concurrent redox reactions of both polythiophene and viologen in solid-state devices and correlate such reactions with device conductivity. The mechanism deduced from spectroscopic and electronic monitoring should guide significant improvements in memory performance.     

Thermochromic Color Switching to Temperature Controlled Volatile Memory and Counter Operations with Metal–Organic Complexes and Hybrid Gels

Temperature is often not considered as a precision stimulus for artificial chemical systems in contrast to the host–guest interactions related to many natural processes. Similarly, mimicking multi‐state volatile memory operations using a single molecular system with temperature as a precision stimulus is highly laborious. Here we demonstrate how a mixture of iron(II) chloride and bipyridine can be used as a reversible color‐to‐colorless thermochromic switch and logic operators. The generality of the approach was illustrated using Co^II and Ni^II salts that resulted in color‐to‐color transitions. DMSO gels of these systems, exhibited reversible opaque‐transparency switching. More importantly, optically readable multi‐state volatile memory with temperature as a precision input has been demonstrated. The stored data is volatile and is lost instantaneously upon withdrawal or change of temperature. Simultaneous read‐out at multiple wavelengths results in single‐input/multi‐output sequential logic operations such as data accumulators (counters) leading to volatile memory states. The present system provides access to thermoresponsive materials wherein temperature can be used as a precision stimulus.     

Design of a hybrid biosensor for enhanced phosphopeptide recognition based on a phosphoprotein binding domain coupled with a fluorescent chemosensor

Protein-based fluorescent biosensors with sufficient sensing specificity are useful analytical tools for detection of biologically important substances in complicated biological systems. Here, we present the design of a hybrid biosensor, specific for a bis-phosphorylated peptide, based on a natural phosphoprotein binding domain coupled with an artificial fluorescent chemosensor. The hybrid biosensor consists of a phosphoprotein binding domain, the WW domain, into which has been introduced a fluorescent stilbazole having Zn(II)-dipicolylamine (Dpa) as a phosphate binding motif. It showed strong binding affinity and high sensing selectivity toward a specific bis-phosphorylated peptide in the presence of various phosphate species such as the monophosphorylated peptide, ATP, and others. Detailed fluorescence titration experiments clearly indicate that the binding-induced fluorescence enhancement and the sensing selectivity were achieved by the cooperative action of both binding sites of the hybrid biosensor, i.e., the WW domain and the Zn(II)-Dpa chemosensor unit. Thus, it is clear that the tethered Zn(II)-Dpa-stilbazole unit operated not only as a fluorescence signal transducer, but also as a sub-binding site in the hybrid biosensor. Taking advantage of its selective sensing property, the hybrid biosensor was successfully applied to real-time and label-free fluorescence monitoring of a protein kinase-catalyzed phosphorylation.     

Development of a Protein Chip to Measure PKCβ Activity

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C β (PKCβ) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCβ activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCβ was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCβ substrate peptide on epoxy-treated slide surface, PKCβ reaction mix was applied over the immobilized MBP-fused PKCβ substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCβ protein chip as a high-throughput screening tool in the screening of depigmenting agents.     

Simultaneously multiply-configurable or superposed molecular logic systems composed of ICT (internal charge transfer) chromophores and fluorophores integrated with one- or two-ion receptors

Integrated "ICT chromophore-receptor" systems show ion-induced shifts in their electronic absorption spectra. The wavelength of observation can be used to reversibly configure the system to any of the four logic operations permissible with a single input (YES, NOT, PASS 1, PASS 0), under conditions of ion input and transmittance output. We demonstrate these with dyes integrated into Tsien's calcium receptor, 1-2. Applying multiple ion inputs to 1-2 also allows us to perform two- or three-input OR or NOR operations. The weak fluorescence output of 1 also shows YES or NOT logic depending on how it is configured by excitation and emission wavelengths. Integrated "receptor(1)-ICT chromophore-receptor(2)" systems 3-5 selectively target two ions into the receptor terminals. The ion-induced transmittance output of 3-5 can also be configured via wavelength to illustrate several logic types including, most importantly, XOR. The opposite effects of the two ions on the energy of the chromophore excited state is responsible for this behaviour. INHIBIT and REVERSE IMPLICATION are two of the other logic types seen here. Integration of XOR logic with a preceding OR operation can be arranged by using three ion inputs. The fluorescence output of these systems can be configured via wavelength to display INHIBIT or NOR logic under two-input conditions. The superposition or multiplicity of logic gate configurations is an unusual consequence of the ability to simultaneously observe multiple wavelengths.     

Specificity analysis-based identification of new methylation targets of the SET7/9 protein lysine methyltransferase

We applied peptide array methylation to determine an optimized target sequence for the SET7/9 (KMT7) protein lysine methyltransferase. Based on this, we identified 91 new peptide substrates from human proteins, many of them better than known substrates. We confirmed methylation of corresponding protein domains in?vitro and in?vivo with a high success rate for strongly methylated peptides and showed methylation of nine nonhistone proteins (AKA6, CENPC1, MeCP2, MINT, PPARBP, ZDH8, Cullin1, IRF1, and [weakly] TTK) and of H2A and H2B, which more than doubles the number of known SET7/9 targets. SET7/9 is inhibited by phosphorylation of histone and nonhistone substrate proteins. One lysine in the MINT protein is dimethylated in?vitro and in?vivo demonstrating that the product pattern created by SET7/9 depends on the amino acid sequence context of the target site.     

Photo- and Thermochromic Spirans. New Metal Chelates Based on Azomethines and Hydrazones Containing a Spiropyran Fragment

Imine- and hydrazone-based metal chelates containing a spiropyran fragment derived from 3-methyl-4-oxo[2H]-1,3-benzoxazinone were prepared. Attempts are made to monitor the photochromic activity of spiropyran using nontraditional -acceptor substituents (products of condensation of the formyl group in 6"-dimethyl-8"-formylspiro-(4-oxo-3,4-dihydro-2H-1,3-benzoxazine-2,2"-[2H]chromene) with o- aminophenol and aromatic acid hydrazides) and using transition metal ions.