A Protein‐Based Pentavalent Inhibitor of the Cholera Toxin B‐Subunit

Protein toxins produced by bacteria are the cause of many life‐threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site‐specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC₅₀) of 104 pM for the CT B‐subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies.     

Effect of TiO2 on Altering Direction of Interfacial Charge Transfer in a TiO2‐Ag‐MPY‐FePc System by SERS

Interfacial charge transfer (CT) is of interest owing to its effect on the performance of molecular photovoltaic (PV) devices. The characteristics and structures of interfacial materials, such as TiO₂ nanoparticles (NPs) in some solar cells, are employed to adjust the CT process. In this study, three kinds of interfacial systems, including a solar cell‐like TiO₂‐Ag‐ p‐mercaptopyridine (MPY)‐ iron phthalocyanine (FePc) system, are compared to investigate the interfacial CT process using surface‐enhanced Raman scattering (SERS) spectroscopy. The SERS results show the significance of TiO₂ NPs in the system on altering the direction and path of the interfacial CT, which is closely associated with the CT enhancement contribution to SERS in such an interfacial system. SERS spectroscopy is expected to be a promising technique for the exploration and estimation of the interfacial CT behavior in PV devices, which may further extend the applications of SERS in the field of solar cells.     

Is it common for charge recombination to be faster than charge separation?

Attaining long-lived charge-transfer (CT) states is of the utmost importance for energy science, photocatalysis, and materials engineering. When charge separation (CS) is slower than consequent charge recombination (CR), formation of a CT state is not apparent, yet the CT process provides parallel pathways for deactivation of electronically excited systems. The nuclear, or Franck-Condon (FC), contributions to the CT kinetics, as implemented by various formalisms based on the Marcus transition-state theory, provide an excellent platform for designing systems that produce long-lived CT states. Such approaches, however, tend to underestimate the complexity of alternative parameters that govern CT kinetics. Here we show a comparative analysis of two systems that have quite similar FC CT characteristics but manifest distinctly different CT kinetics. A decrease in the donor-acceptor electronic coupling during the charge-separation step provides an alternative route for slowing down undesired charge recombination. These examples suggest that, while infrequently reported and discussed, cases where CR is faster than CS are not necessarily rare occurrences.     

QM/MM Investigations on the Bioluminescent Decomposition of Coelenterazine Dioxetanone in Obelin

The bioluminescent mechanism of colenterazine dioxetanone(CZD) in the photoprotein of Obelia(obelin) was investigated by the combined quantum and molecular mechanics(QM/MM) method at TD-DFT level, which involved the real protein environment in decomposition of 1,2-dioxetanones. The anionic decomposition of CZD in (CZD+H₂O)^- model can go through a charge transfer(CT) catalyzed asynchronous-concerted process, which can be elucidated by the gradual reversible CT initiated luminescence(GRCTIL) mechanism. The neutral CZD in (CZDH+H₂O) decomposes through an uncatalyzed non-CT biradical process. The anionic decomposition catalyzed by CT, in which the S₀/S₁ surface "double crossing" hence has ability to provide high quantum yield of singlet chemiexcitation is thus more possible in bioluminescence of photoprotein.     

Mechanistic insights in charge-transfer-induced luminescence of 1,2-dioxetanones with a substituent of low oxidation potential

We have investigated the decomposition pathway of dioxetanones 1c with a phenoxide anion group by the B3LYP/6-31+G(d) method together with the second-order multireference M?ller-Plesset perturbation (MRMP) theory and propose charge-transfer-induced luminescence (CTIL) with polarization-induced branching excitation processes. In the gas phase, the thermal decomposition of 1c occurs by an asynchronous two-stage pathway without a discrete intermediate; that is, the initial O-O bond breaking to generate a charge-transfer (CT) diradical species is immediately followed by the subsequent C-C bond breaking with simultaneous back CT, which is responsible for the surface crossing at the avoided crossing. The activation energy is dramatically reduced from 19.4 to 3.8 kcal mol(-)(1) by the deprotonation of phenol meta-1d to its anion meta-1c, showing an important role of the endothermic CT. The odd/even selection rule for the chemiluminescence efficiency can be explained by the orbital interaction for the back CT between the carbonyl pi orbital and either a HOMO or a LUMO of the generated light emitters. To examine the accessibility of the chemically initiated electron exchange luminescence (CIEEL) route, we considered the solvent effects on the free-energy change of meta-1c by using continuum solvent models. The bending vibration mode of the CO(2) fragment is specifically considered. Borderline features emerges from the solution-phase CT reaction of meta-1c, which depends on the solvent polarity: one is a nonadiabatic or adiabatic back CT process (polarization-induced concerted CTIL), and the other is a radical dissociation, i.e., complete one-electron-transfer process (CIEEL).     

SPAP2基因的克隆、融合表达及分离纯化

本文构建的表达载体pGex-2T-SPAP2CT在大肠杆菌中表达出可融性蛋白, 其分子量为46 000, 经纯化后得到产率为10%、 纯度大于90%的GST-SPAP2CT蛋白.     

Differential proteome analysis of tonsils from children with chronic tonsillitis or with hyperplasia reveals disease-associated protein expression differences

A proteomic approach has been used to establish a proteome map and differentiate between the protein composition of tonsils from patients with chronic tonsillitis (CT) and that of tonsils with hyperplasia (HPL). Two-dimensional gel analysis was performed with material from four patients with HPL and five patients with CT. An average of approximately 600 spots were detected in each gel. A total of 127 different proteins were identified in 158 spots analyzed by mass spectrometry. Our study revealed disease-associated differences between protein abundance for two protein spots, an HSP27 isoform and UMP-CMP kinase. Both protein spots were more abundant in the CT group. HSP27 ELISA was performed for 32 patients, 12 belonging to the HPL group and 20 to the CT group. ELISA could not be used to differentiate HSP27 isoforms nor to distinguish CT from HPL. HSP27 was found to migrate to two further protein spots in the 2D gels. The differently expressed HSP27 isoform migrated as the most acidic of all the HSP27 isoforms detected, indicating the highest degree of phosphorylation. The sum of all three HSP27 abundances in the gels from the CT group was not different from that of the HPL group, consistent with the ELISA results. Our results suggest that phosphorylation differences caused the observed migration differences of HSP27. Together with the UMP-CMP kinase abundance differences, we conclude that kinase and/or phosphatase activity are different in CT and HPL. This paper was presented at the 38th Annual Meeting of the German Society for Mass Spectrometry (DGMS) held in March 2005 in Rostock, Germany.     

Charge transfer between DNA and proteins in the nucleosomes

Recently X-ray diffraction provided the structure of nucleosomes. External disturbances can unwrap DNA from the histone–protein and their genetic information becomes readable. This is strongly connected with cancer initiation. Therefore, first we performed charge transfer (CT) calculations between polythymidine and a periodic model-protein chain with a lysine or arginine and three glycines. The CT calculations were repeated between the infinite chains using combined solid state physical and quantum chemical methods. We found that the CT between the unit cells of an infinite polythymidine and poly(lysine-triglycine) is 0.04 e and 0.03 e for poly(arginine-triglycine). We investigated the influence of the basis set quality on the calculated CT values using a molecular model built of a thymidine and lysine or arginine. We have calculated also the bands of polythymidine and the two protein model chains. We have found that the differences between the highest level of the valence band of single polythymidine chain and the lowest level of the conduction bands of the model protein chains (6-11 eV depending on the basis set) are too large to assume a direct CT between these two bands.     

Exciton-coupled charge-transfer dynamics in a porphyrin J-aggregate/TiO(2) complex

Exciton-coupled charge-transfer (CT) dynamics in TiO(2) nanoparticles (NP) sensitized with porphyrin J-aggregates has been studied by femtosecond time-resolved transient absorption spectroscopy. J-aggregates of 5,10,15-triphenyl-20-(3,4-dihydroxyphenyl) porphyrin (TPPcat) form CT complexes on TiO(2) NP surfaces. Catechol-mediated strong CT coupling between J-aggregate and TiO(2) NP facilitates interfacial exciton dissociation for electron injection into the conduction band of the TiO(2) nanoparticle in pulse width limited time (<80 fs). Here, the electron-transfer (<80 fs) process dominates over the intrinsic exciton-relaxation process (J-aggregates: ca. 200 fs) on account of exciton-coupled CT interaction. The parent hole on J-aggregates is delocalized through J-aggregate excitonic coherence. As a result, holes immobilized on J-aggregates are spatially less accessible to electrons injected into TiO(2) , and thus the back electron transfer (BET) process is slower than that of the monomer/TiO(2) system. The J-aggregate/porphyrin system shows exciton spectral and temporal properties for better charge separation in strongly coupled composite systems.     

A Quantitative View of Charge Transfer in the Hydrogen Bond: The Water Dimer Case

The hydrogen bond represents a fundamental intermolecular interaction that binds molecules in vapor and liquid water. A crucial and debated aspect of its electronic structure and chemistry is the charge transfer (CT) accompanying it. Much effort has been devoted, in particular, to the study of the smallest prototype system, the water dimer, but even here results and interpretations differ widely. In this paper, we reassess CT in the water dimer by using charge‐displacement analysis. Besides a reliable estimate of the amount of CT (14.6 me) that characterizes the system, our study provides an unambiguous context, and very useful bounds, within which CT effects may be evaluated, crucially including the associated energy stabilization.     

Targeted Degradation of PD-L1 and Activation of the STING Pathway by Carbon-Dot-Based PROTACs for Cancer Immunotherapy

Proteolysis targeting chimeras (PROTACs) technology is an emerging approach to degrade disease-associated proteins. Here, we report carbon-dot (CD)-based PROTACs (CDTACs) that degrade membrane proteins via the ubiquitin-proteasome system. CDTACs can bind to programmed cell death ligand 1 (PD-L1), recruit cereblon (CRBN) to induce PD-L1 ubiquitination, and degrade them with proteasomes. Fasting-mimicking diet (FMD) is also used to enhance the cellular uptake and proteasome activity. More than 99 % or 90 % of PD-L1 in CT26 or B16-F10 tumor cells can be degraded by CDTACs, respectively. Furthermore, CDTACs can activate the stimulator of interferon genes (STING) pathway to trigger immune responses. Thus, CDTACs with FMD treatment effectively inhibit the growth of CT26 and B16-F10 tumors. Compared with small-molecule-based PROTACs, CDTACs offer several advantages, such as efficient membrane protein degradation, targeted tumor accumulation, immune system activation, and in vivo detection.     

Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot

This paper describes the application of an automated size‐based capillary Western blot system (Sally instrument) from ProteinSimple, Inc., for biopharmaceutical fusion‐Fc protein characterization and evaluation of its purification process. The fusion‐Fc protein column purification from an excess of single chain Fc polypeptide and removal of an enzyme coexpressed for protein maturation have been demonstrated using an automated capillary Western system. The clearance of a selected host cell protein (HCP) present in cell culture of fusion‐Fc protein was also quantitatively monitored throughout the protein purification process. Additionally, the low levels of fusion‐Fc product‐related impurities detected by traditional slab gel Western blot were confirmed by the automated capillary Western system. Compared to the manual approach, the automated capillary Western blot provides the advantages of ease of operation, higher sample throughput, greater linearity range, and higher precision for protein quantitation.     

Real-time Quantitative RT-PCR for CT9 Level in Human Cancer

Introduction Malignantdiseasesarecharacterizedbytheunreg ulatedgrowthoftransformedcells.Inrecentyears,dramaticinsightsintothemolecularmechanismsofthis phenomenonhavebeenachievedfrombasiccancerre search.Manycellularfunctionsareregulatedbychan gesingeneexpr…     

Femtosecond dynamics after ionization: 2-phenylethyl-N,N-dimethylamine as a model system for nonresonant downhill charge transfer in peptides

The cation of 2-phenylethyl-N,N-dimethylamine (PENNA) offers two local sites for the charge: the amine group and 0.7 eV higher in energy the phenyl chromophore. In this paper, we investigate the dynamics of the charge transfer (CT) from the phenyl to the amine site. We present a femtosecond resonant two-color photoionization spectrum which shows that the femtosecond pump laser pulse is resonant in the phenyl chromophore. As shown previously with resonant wavelengths the aromatic phenyl chromophore can be then selectively ionized. Because the state "charge in the phenyl chromophore" is the first excited state in the PENNA cation, it can relax to the lower-energetic state "charge in the amine site". To follow this CT dynamics, femtosecond probe photoabsorption of green light (vis) is used. The vis light is absorbed by the charged phenyl chromophore, but not by the neutral phenyl and the neutral or cationic amine group. Thus, the absorption of vis photons of the probe laser pulse is switched off by the CT process. For detection of the resonant absorption of two or more vis photons in the cation the intensity of a fragmentation channel is monitored which opens only at high internal energy. The CT dynamics in PENNA cations has a time constant of 80 +/- 28 fs and is therefore not a purely electronic process. Because of its structural similarity to phenylalanine, PENNA is a model system for a downhill charge transfer in peptide cations.     

Amine‐Responsive Adaptable Nanospaces: Fluorescent Porous Coordination Polymer for Molecular Recognition

Flexible and dynamic porous coordination polymers (PCPs) with well‐defined nanospaces composed of chromophoric organic linkers provide a scaffold for encapsulation of versatile guest molecules through noncovalent interactions. PCPs thus provide a potential platform for molecular recognition. Herein, we report a flexible 3D supramolecular framework {[Zn(ndc)(o‐phen)]?DMF}_n (o‐phen=1,10‐phenanthroline, ndc=2,6‐napthalenedicarboxylate) with confined nanospaces that can accommodate different electron‐donating aromatic amine guests with selective turn‐on emission signaling. This system serves as a molecular recognition platform through an emission‐readout process. Such unprecedented tunable emission with different amines is attributed to its emissive charge‐transfer (CT) complexation with o‐phen linkers. In certain cases this CT emission is further amplified by energy transfer from the chromophoric linker unit ndc, as evidenced by single‐crystal X‐ray structural characterization.     

Roles of p97-Associated Deubiquitinases in Protein Quality Control at the Endoplasmic Reticulum

To maintain protein homeostasis in the ER, an ER protein quality control system retains unfolded polypeptides and misassembled membrane proteins, allowing only properly folded proteins to exit the ER. Misfolded proteins held in the ER are retrotranslocated into the cytosol, ubiquitinated, and degraded by the proteasome through the ER-associated degradation pathway (ERAD). By timely eliminating misfolded proteins, the ERAD system alleviates cytotoxic stress imposed by protein misfolding. It is well established that ER-associated ubiquitin ligases play pivotal roles in ERAD by assembling ubiquitin conjugates on retrotranslocation substrates, which serve as degradation signals for the proteasome. Surprisingly, recent studies have revealed an equally important function for deubiquitinases (DUBs), enzymes that disassemble ubiquitin chains, in ERAD. Intriguingly, many ERAD specific DUBs are physically associated with the retrotranslocation- driving ATPase p97. Here we discuss the potential functions of p97-associated DUBs including ataxin-3 and YOD1. Our goal is to integrate the emerging evidence into models that may explain how protein quality control could benefit from deubiquitination, a process previously deemed destructive for proteasomal degradation.     

Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags

High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Coli cell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes approximately 85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device.     

Frame‐Guided Assembly of Vesicles with Programmed Geometry and Dimensions

In molecular self‐assembly molecules form organized structures or patterns. The control of the self‐assembly process is an important and challenging topic. Inspired by the cytoskeletal‐membrane protein lipid bilayer system that determines the shape of eukaryotic cells, we developed a frame‐guided assembly process as a general strategy to prepare heterovesicles with programmed geometry and dimensions. This method offers greater control over self‐assembly which may benefit the understanding of the formation mechanism as well as the functions of the cell membrane.     

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb5 molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.