Miwako Asanuma

Turn-ON fluorescent affinity labeling using a small bifunctional O-nitrobenzoxadiazole unit

Affinity labeling has become a powerful tool to identify target proteins, as well as to visualize/characterize target functions in living cells. However, although various functional groups have been utilized for affinity labeling, many of them have disadvantages such as complex structure and large size. To address this problem, we designed a simple chemical probe bearing a small bifunctional O-NBD unit (NBD: nitrobenzoxadiazole). Model ligand–protein experiments showed that the O-NBD unit has excellent characteristics for target-specific labeling even in the presence of a large excess of non-target proteins. Moreover, attachment of the O-NBD unit to N,N-dialkyl-2-phenylindol-3-ylglyoxylamides (PIGAs), which are recently developed translocator protein (TSPO) ligands, enabled us to visualize mitochondria expressing TSPO in living cells by means of “turn-ON” fluorescence. Two-dimensional PAGE analysis of the labeled mouse kidney mitochondria strongly suggested that our synthetic probe selectively modified a partner protein of TSPO, voltage-dependent anion channel (VDAC).

VDAC3 gating is activated by suppression of disulfide-bond formation between the N-terminal region and the bottom of the pore

The voltage-dependent anion channels (VDACs), VDAC1, VDAC2, and VDAC3, are pore-forming proteins that control metabolite flux between mitochondria and cytoplasm. VDAC1 and VDAC2 have voltage-dependent gating activity, whereas VDAC3 is thought to have weak activity. The aim of this study was to analyze the channel properties of all three human VDAC isoforms and to clarify the channel function of VDAC3. Bacterially expressed recombinant human VDAC proteins were reconstituted into artificial planar lipid bilayers and their gating activities were evaluated. VDAC1 and VDAC2 had typical voltage-dependent gating activity, whereas the gating of VDAC3 was weak, as reported. However, gating of VDAC3 was evoked by dithiothreitol (DTT) and S-nitrosoglutathione (GSNO), which are thought to suppress disulfide-bond formation. Several cysteine mutants of VDAC3 also exhibited typical voltage-gating. Our results indicate that channel gating was induced by reduction of a disulfide-bond linking the N-terminal region to the bottom of the pore. Thus, channel gating of VDAC3 might be controlled by redox sensing under physiological conditions.

Alkyne-Tag SERS Screening and Identification of Small-Molecule-Binding Sites in Protein

Identification of small-molecule-binding sites in protein is important for drug discovery and analysis of protein function. Modified amino-acid residue(s) can be identified by proteolytic cleavage followed by liquid chromatography-mass spectrometry (LC-MS), but this is often hindered by the complexity of the peptide mixtures. We have developed alkyne-tag Raman screening (ATRaS) for identifying binding sites. In ATRaS, small molecules are tagged with alkyne and form covalent bond with proteins. After proteolysis and HPLC, fractions containing the labeled peptides with alkyne tags are detected by means of surface-enhanced Raman scattering (SERS) using silver nanoparticles and sent to MS/MS to identify the binding site. The use of SERS realizes high sensitivity (detection limit: ∼100 femtomole) and reproducibility in the peptide screening. By using an automated ATRaS system, we successfully identified the inhibitor-binding site in cysteine protease cathepsin B, a potential drug target and prognostic marker for tumor metastasis. We further showed that the ATRaS system works for complex mixtures of trypsin-digested cell lysate. The ATRaS technology, which provides high molecular selectivity to LC-MS analysis, has potential to contribute in various research fields, such as drug discovery, proteomics, metabolomics and chemical biology.

NTA-mediated protein capturing strategy in screening experiments for small organic molecules by surface plasmon resonance

Nitrilotriacetate (NTA)‐mediated capture of a histidine‐tagged protein is widely used as an easy and simple method to reversibly immobilize the protein onto a sensor chip for surface plasmon resonance (SPR). However, in spite of its advantages, the NTA‐capturing strategy is rarely employed for ligand screening experiments using SPR, because it was thought to cause substantial errors in binding responses, due to the inevitable protein dissociation during the monitoring period. In this study, as demonstrated in a ligand screening for the histidine‐tagged SH3 domain of the human phosphatidylinositol 3‐kinase p85α subunit, false responses after adhesion of undesirable compounds to a target protein could be minimized with the NTA strategy, while binding responses of a positive control peptide still stayed within a 1%‐deviation against the theoretical binding capacity.

Solid-state 17 O NMR Study of Small Biological Compounds

We present a systematic experimental and theoretical investigation of the oxygen chemical shielding and electric-field-gradient tensors in polycrystalline amino acids and a peptide. Analysis of the 17O magic-angle-spinning (MAS), multiple-quantum MAS, and stationary nuclear magnetic resonance (NMR) spectra yield the magnitudes and the relative orientations between the two NMR tensors. The obtained 17O NMR parameters are sensitive to the hydrogen bond environments. We also demonstrate that solid-state 17O NMR is potentially useful for studying the secondary structures of peptides and proteins.

CDC25A-inhibitory RE derivatives bind to pocket adjacent to the catalytic site.

RE derivatives, which are cell-permeable and non-electrophilic dual-specificity protein phosphatase inhibitors developed in our laboratory, inhibit CDC25A/B non-competitively, as determined by means of kinetic experiments. To identify the binding site of RE derivatives, we designed and synthesized the new probe molecule RE142, having a Michael acceptor functionality for covalent bond formation with the enzyme, a biotin tag to enable enrichment of probe-bound peptide(s), and a chemically cleavable linker to facilitate release of probe-bound peptides from avidin beads. LC-MS analysis indicated that RE142 binds to one of the residues Cys384-Tyr386 of CDC25A, within a pocket adjacent to the catalytic site.