Analysis of Cell-Surface Receptor Dynamics through Covalent Labeling by Catalyst-Tethered Antibody

A general technique for introducing biophysical probes into selected receptors in their native environment is valuable for the study of their structure, dynamics, function, and molecular interactions. A number of such techniques rely on genetic engineering, which is not applicable for the study of endogenous proteins, and such approaches often suffer from artifacts due to the overexpression and bulky size of the probes/protein tags used. Here we designed novel catalyst-antibody conjugates capable of introducing small chemical probes into receptor proteins such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in a selective manner on the surface of living cells. Because of the selectivity and efficiency of this labeling technique, we were able to monitor the cellular dynamics and lifetime of HER2 endogenously expressed on cancer cells. More significantly, the current labeling technique comprises a stable covalent bond, which combined with a peptide mass fingerprinting analysis allowed epitope mapping of antibodies on living cells and identification of potential binding sites of anti-EGFR affibody. Although as yet unreported in the literature, the binding sites predicted by our labeling method were consistently supported by the subsequent mutation and binding assay experiments. In addition, this covalent labeling method provided experimental evidence that HER2 exhibits a more dynamic structure than expected on the basis of crystallographic analysis alone. Our novel catalyst-antibody conjugates are expected to provide a general tool for investigating the protein trafficking, fluctuation, and molecular interactions of an important class of cell-surface receptors on live cell surfaces

Kinetic Study of the 7-<i>endo</i> Selective Radical Cyclization of <i>N</i>-<i>tert</i>-Butyl‑<i>o</i>‑bromobenzylmethacryl Amides: Kinetic Investigation of the Cyclization and 1,7-Hydrogen Transfer of Aromatic Radicals

A kinetic investigation of the radical cyclization of <i>N</i>-<i>tert</i>-butyl-<i>o</i>-bromobenzyl­methacryl amides to give 2-benzazepines via 7-<i>endo</i> selective cyclization was undertaken. The aryl radical generated from the amide precursor by treatment with Bu₃SnH gave the three compounds, which are a 7-<i>endo</i> cyclized adduct, a 6-exocyclized adduct, and a reduced product. The cyclization reactions under various Bu₃SnH concentrations were traced by GC analysis. The 7-<i>endo</i>/6-<i>exo</i> selectivity was constant irrespective of variation in Bu₃SnH concentration. These results revealed that regioselectivity is controlled in a kinetic manner and that there is no possibility of a neophyl rearrangement. The use of Bu₃SnD revealed that 1,7-hydrogen transfer, in which an aryl radical abstracts a hydrogen atom from the methallylic methyl group, occurs during the reaction. Hydrogen abstraction from toluene, the reaction solvent, was also observed. The 1,7-transfer rate depended on the Bu₃SnX (X = H or D), and the reaction kinetics was examined. The <i>k</i>_H/<i>k</i>_D value for the hydrogen abstraction of aryl radical from Bu₃SnX (X = H or D) was estimated using 4-bromoanisol. The utilization of these values revealed the overall reaction kinetics and relative rates for the cyclization and reduction by Bu₃SnX (X = H or D). Kinetic parameters for hydrogen abstraction from toluene by aryl radicals were also estimated

The Production of Nitrous Oxide by the Heme/Nonheme Diiron Center of Engineered Myoglobins (Fe_BMbs) Proceeds through a <i>trans</i>-Iron-Nitrosyl Dimer

Denitrifying NO reductases are transmembrane protein complexes that are evolutionarily related to heme/copper terminal oxidases. They utilize a heme/nonheme diiron center to reduce two NO molecules to N₂O. Engineering a nonheme Fe_B site within the heme distal pocket of sperm whale myoglobin has offered well-defined diiron clusters for the investigation of the mechanism of NO reduction in these unique active sites. In this study, we use FTIR spectroscopy to monitor the production of N₂O in solution and to show that the presence of a distal Fe_B^II is not sufficient to produce the expected product. However, the addition of a glutamate side chain peripheral to the diiron site allows for 50% of a productive single-turnover reaction. Unproductive reactions are characterized by resonance Raman spectroscopy as dinitrosyl complexes, where one NO molecule is bound to the heme iron to form a five-coordinate low-spin {FeNO}⁷ species with ν­(FeNO)_heme and ν­(NO)_heme at 522 and 1660 cm^–1, and a second NO molecule is bound to the nonheme Fe_B site with a ν­(NO)_FeB at 1755 cm^–1. Stopped-flow UV–vis absorption coupled with rapid-freeze-quench resonance Raman spectroscopy provide a detailed map of the reaction coordinates leading to the unproductive iron-nitrosyl dimer. Unexpectedly, NO binding to Fe_B is kinetically favored and occurs prior to the binding of a second NO to the heme iron, leading to a (six-coordinate low-spin heme-nitrosyl/Fe_B-nitrosyl) transient dinitrosyl complex with characteristic ν­(FeNO)_heme at 570 ± 2 cm^–1 and ν­(NO)_FeB at 1755 cm^–1. Without the addition of a peripheral glutamate, the dinitrosyl complex is converted to a dead-end product after the dissociation of the proximal histidine of the heme iron, but the added peripheral glutamate side chain in Fe_BMb2 lowers the rate of dissociation of the promixal histidine which in turn allows the (six-coordinate low-spin heme-nitrosyl/Fe_B-nitrosyl) transient dinitrosyl complex to decay with production of N₂O at a rate of 0.7 s^–1 at 4 °C. Taken together, our results support the proposed trans mechanism of NO reduction in NORs

Electrochemical Fine Tuning of the Plasmonic Properties of Au Lattice Structures

We tuned the plasmonic properties of the Au lattice structure by electrochemical potential control. Au lattice structures with different values of the spacing, diameter, and height show characteristic optical properties determined by the surface lattice resonance of the localized surface plasmon mode. Electrochemical potential control can change the metal structures through metal dissolution, as well as the energy of the electrons in metals. In situ real time observation of the optical properties of Au lattice structures by electrochemical dark-field scattering microscopy shows the fine-tuning of the plasmonic properties with characteristic resonance energy and controlled spectral width. By controlling surface dissolution of the Au lattice structure at a rate of a few nanometers per minute, we tuned the plasmonic properties and achieved a spectral width of 0.145 eV at a maximum resonance of 1.74 eV (714 nm)

Vibrational Analysis of Mononitrosyl Complexes in Hemerythrin and Flavodiiron Proteins: Relevance to Detoxifying NO Reductase

Flavodiiron proteins (FDPs) play important roles in the microbial nitrosative stress response in low-oxygen environments by reductively scavenging nitric oxide (NO). Recently, we showed that FMN-free diferrous FDP from <i>Thermotoga maritima</i> exposed to 1 equiv NO forms a stable diiron-mononitrosyl complex (deflavo-FDP­(NO)) that can react further with NO to form N₂O [Hayashi, T.; Caranto, J. D.; Wampler, D. A; Kurtz, D. M., Jr.; Moënne-Loccoz, P. Biochemistry 2010, 49, 7040−7049]. Here we report resonance Raman and low-temperature photolysis FTIR data that better define the structure of this diiron-mononitrosyl complex. We first validate this approach using the stable diiron-mononitrosyl complex of hemerythrin, Hr­(NO), for which we observe a ν­(NO) at 1658 cm^–1, the lowest ν­(NO) ever reported for a nonheme {FeNO}⁷ species. Both deflavo-FDP­(NO) and the mononitrosyl adduct of the flavinated FPD (FDP­(NO)) show ν­(NO) at 1681 cm^–1, which is also unusually low. These results indicate that, in Hr­(NO) and FDP­(NO), the coordinated NO is exceptionally electron rich, more closely approaching the Fe­(III)­(NO^–) resonance structure. In the case of Hr­(NO), this polarization may be promoted by steric enforcement of an unusually small FeNO angle, while in FDP­(NO), the Fe­(III)­(NO^–) structure may be due to a semibridging electrostatic interaction with the second Fe­(II) ion. In Hr­(NO), accessibility and steric constraints prevent further reaction of the diiron-mononitrosyl complex with NO, whereas in FDP­(NO) the increased nucleophilicity of the nitrosyl group may promote attack by a second NO to produce N₂O. This latter scenario is supported by theoretical modeling [Blomberg, L. M.; Blomberg, M. R.; Siegbahn, P. E. J. Biol. Inorg. Chem. 2007, 12, 79−89]. Published vibrational data on bioengineered models of denitrifying heme-nonheme NO reductases [Hayashi, T.; Miner, K. D.; Yeung, N.; Lin, Y.-W.; Lu, Y.; Moënne-Loccoz, P. Biochemistry 2011, 50, 5939−5947] support a similar mode of activation of a heme {FeNO}⁷ species by the nearby nonheme Fe­(II)

Semisynthetic Lectin–4-Dimethylaminopyridine Conjugates for Labeling and Profiling Glycoproteins on Live Cell Surfaces

Glycoproteins on cell surfaces play important roles in biological processes, including cell–cell interaction/signaling, immune response, and cell differentiation. Given the diversity of the structure of glycans, labeling and imaging of selected glycoproteins are challenging, although several promising strategies have been developed recently. Here, we design and construct semisynthetic reactive lectins (sugar-binding proteins) that are able to selectively label glycoproteins. Congerin II, an animal galectin, and wheat germ agglutinin are conjugated with 4-dimethylaminopyridine (DMAP), a well-known acyl transfer catalyst by our affinity-guided DMAP method and Cu­(I)-assisted click chemistry. Selective labeling of glycoproteins is facilitated by the DMAP-tethered lectin catalysts both <i>in vitro</i> and on living cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis enabled us to isolate labeled glycoproteins that are uniquely exposed on distinct cell lines. Furthermore, the combination of immunoprecipitation with mass spectrometry (MS)-fingerprinting techniques allowed us to characterize 48 glycoproteins endogenously expressed on HeLa cells, and some low-abundant glycoproteins, such as epidermal growth factor receptor (EGFR) and neuropilin-1, were successfully identified. Our results demonstrate that semisynthetic DMAP-tethered lectins provide a new tool for labeling and profiling glycoproteins on living cells

Vapochromic Luminescence and Flexibility Control of Porous Coordination Polymers by Substitution of Luminescent Multinuclear Cu(I) Cluster Nodes

Two luminescent porous coordination polymers (PCPs), i.e., [Cu₂(μ₂-I)₂ctpyz]_<i>n</i> and [Cu₄(μ₃-I)₄ctpyz]_<i>n</i> (<b>Cu2</b> and <b>Cu4</b>, respectively; ctpyz = <i>cis</i>-1,3,5-cyclohexanetriyl-2,2′,2″-tripyrazine), were successfully synthesized and characterized by single-crystal X-ray diffraction and luminescence spectroscopic measurements. <b>Cu2</b> consists of rhombus-type dinuclear {Cu₂I₂} cores bridged by ctpyz ligands, while <b>Cu4</b> is constructed of cubane-type tetranuclear {Cu₄I₄} cores bridged by ctpyz ligands. The void fraction of <b>Cu4</b> is estimated to be 48.0%, which is significantly larger than that of <b>Cu2</b> (19.9%). Under UV irradiation, both PCPs exhibit red luminescence at room temperature in the solid state (λ_em values of 660 and 614 nm for <b>Cu2</b> and <b>Cu4</b>, respectively). Although the phosphorescence of <b>Cu2</b> does not change upon removal and/or adsorption of EtOH solvent molecules in the porous channels, the solid-state emission maximum of <b>Cu4</b> red-shifts by 36 nm (λ_em = 650 nm) upon the removal of the adsorbed benzonitrile (PhCN) molecules from the porous channels (and vice versa). This large difference in the vapochromic behavior of <b>Cu2</b> and <b>Cu4</b> is closely related to the framework flexibility. The framework of <b>Cu2</b> is sufficiently rigid to retain the porous structure without solvated EtOH molecules, whereas the porous structure of <b>Cu4</b> collapses easily after removal of the adsorbed PhCN molecules to form a nonporous amorphous phase. The original vapor-adsorbed porous structure of <b>Cu4</b> is regenerated by exposure of the amorphous solid to not only PhCN vapor but also tetrahydrofuran, acetone, ethyl acetate, and <i>N</i>,<i>N</i>-dimethylformamide vapors. The <b>Cu4</b> structures with the various adsorbed solvents showed almost the same emission maxima as the original PhCN-adsorbed <b>Cu4</b>, except for DMF-adsorbed <b>Cu4</b>, which showed no luminescence probably because of weak coordination of the DMF vapor molecules to the Cu­(I) centers of the tetranuclear {Cu₄I₄} core

Additional file 1 of Factors associated with physical activity following total knee arthroplasty for knee osteoarthritis: a longitudinal study

Supplementary Material

Light-Induced N₂O Production from a Non-heme Iron–Nitrosyl Dimer

Two non-heme iron–nitrosyl species, [Fe₂(<i>N</i>‑Et‑HPTB)­(O₂CPh)­(NO)₂]­(BF₄)₂ (<b>1a</b>) and [Fe₂(<i>N</i>‑Et‑HPTB)­(DMF)₂(NO)­(OH)]­(BF₄)₃ (<b>2a</b>), are characterized by FTIR and resonance Raman spectroscopy. Binding of NO is reversible in both complexes, which are prone to NO photolysis under visible light illumination. Photoproduction of N₂O occurs in high yield for <b>1a</b> but not <b>2a</b>. Low-temperature FTIR photolysis experiments with <b>1a</b> in acetonitrile do not reveal any intermediate species, but in THF at room temperature, a new {FeNO}⁷ species quickly forms under illumination and exhibits a ν­(NO) vibration indicative of nitroxyl-like character. This metastable species reacts further under illumination to produce N₂O. A reaction mechanism is proposed, and implications for NO reduction in flavo­diiron proteins are discussed