Long-term observation of fluorescence of free single molecules to explore protein-folding energy landscapes

A method was developed to detect fluorescence intensity signals from single molecules diffusing freely in a capillary cell. A unique optical system based on a spherical mirror was designed to enable quantitative detection of the fluorescence intensity. Furthermore, "flow-and-stop" control of the sample can extend the observation time of single molecules to several seconds, which is more than 1000 times longer than the observation time available using a typical confocal method. We used this method to scrutinize the fluorescence time series of the labeled cytochrome c in the unfolded state. Time series analyses of the trajectories based on local equilibrium state analysis revealed dynamically differing substates on a millisecond time scale. This system presents a new avenue for experimental characterization of the protein-folding energy landscape.     

Quantitative Assessment of Protein Structural Models by Comparison of H/D Exchange MS Data with Exchange Behavior Accurately Predicted by DXCOREX

Peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) data are often used to qualitatively support models for protein structure. We have developed and validated a method (DXCOREX) by which exchange data can be used to quantitatively assess the accuracy of three-dimensional (3-D) models of protein structure. The method utilizes the COREX algorithm to predict a protein’s amide hydrogen exchange rates by reference to a hypothesized structure, and these values are used to generate a virtual data set (deuteron incorporation per peptide) that can be quantitatively compared with the deuteration level of the peptide probes measured by hydrogen exchange experimentation. The accuracy of DXCOREX was established in studies performed with 13 proteins for which both high-resolution structures and experimental data were available. The DXCOREX-calculated and experimental data for each protein was highly correlated. We then employed correlation analysis of DXCOREX-calculated versus DXMS experimental data to assess the accuracy of a recently proposed structural model for the catalytic domain of a Ca²⁺-independent phospholipase A₂. The model’s calculated exchange behavior was highly correlated with the experimental exchange results available for the protein, supporting the accuracy of the proposed model. This method of analysis will substantially increase the precision with which experimental hydrogen exchange data can help decipher challenging questions regarding protein structure and dynamics.     

Wavelength-conversion type picosecond optical switching using a waveguide QPM-SHG/DFG device

We report picosecond all-optical switching in optical communication band using a LiNbO₃ waveguide quasi-phase matched second-harmonic generation/difference-frequency generation device. Analysis based on the beam propagation method showed that 1 ps switching with 3.1% efficiency is feasible with 10 W peak control pulse power. Switching efficiency of ?22 dB was demonstrated using control pulses of 10 ps width and 2 W peak power.     

Heterologous Expression,Biosynthetic Studies,and Ecological Function of the Selective Gq‐Signaling Inhibitor FR900359

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq‐related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC‐1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.     

Aerobic Photooxidative Synthesis of β‐Alkoxy Monohydroperoxides Using an Organo Photoredox Catalyst Controlled by a Base

Transition‐metal‐free synthesis of β‐alkoxy monohydroperoxides via aerobic photooxidation using an acridinium photocatalyst was developed. This method enables the synthesis of some novel hydroperoxides. The peroxide source is molecular oxygen, which is cost‐effective and atomically efficient. Magnesium oxide plays an important role as a base in the catalytic system.     

Voltammetric response of phenylboronic acid monolayer-modified gold electrode to sugars.

The surface of a gold (Au) disk electrode was modified with a self-assembled monolayer consisting of phenylboronic acid moiety to fabricate a voltammetric sensor sensitive to sugars. The modified Au electrode exhibited a voltammetric response to sugars in the presence of Fe(CN)6(3-) ion in the sample solution at neutral pH. The peak current of the cyclic voltammograms decreased depending on the type and concentration of sugars. The dynamic range of the electrode is 3 - 100 mM for glucose and mannose and 1 - 30 mM for fructose. The sugar sensor can be used repeatedly after rinsing in 10 mM acetate buffer (pH 4.5).     

One-pot assembly of tricyclo[6.2.1.0(1,6)]undecan-4-one and related polycyclic compounds by tandem electroreductive cyclization

[reaction: see text] Electroreductive tandem cyclization of 4-allyl-4-(2-bromoprop-2-en-1-yl)cyclohex-2-en-1-one to tricyclo[6.2.1.0(1,6)]undecan-4-one has been demonstrated. This protocol represents an attractive alternative to conventional tandem radical cyclization.     

Synthesis and development of biologically active fluorescent-labeled vitamin K analogues and monitoring of their subcellular distribution

New fluorescent analogues of menaquinone-4 and phylloquinone were prepared and their subcellular distribution monitored using a confocal laser scanning microscope. These analogues incorporate an FITC group anchored to the naphthoquinone skeleton through an amide bond expected to be resistant to metabolism. On their addition to the culture medium, fluorescence was readily observed inside a human osteosarcoma cell line. This result indicates that the fluorescent analogues penetrate into cells the same as vitamin K, and therefore, would be useful for achieving insight into the action mechanism of vitamin K.