Ryo Iizuka

Ryo Iizuka
The University of Tokyo | Todai · Department of Biological Sciences

Doctor of Engineering

About

116
Publications
3,904
Reads
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1,170
Citations
Additional affiliations
March 2009 - March 2020
The University of Tokyo
Position
  • Professor (Assistant)
April 2006 - February 2009
May 2005 - March 2006
Tokyo University of Agriculture and Technology
Position
  • Professor (Assistant)
Education
April 2002 - September 2004
April 2001 - March 2002
April 1997 - March 2001

Publications

Publications (116)
Article
In vitro compartmentalization (IVC) is a method to link genotype and phenotype by confining DNA and an in vitro gene expression system in cell-like compartments such as water-in-oil microdroplets. IVC provides a flexible platform for the selection and directed evolution of peptides, proteins, and RNAs with the desired catalytic, binding, and regula...
Article
Single-molecule technologies can provide detailed information regarding molecular mechanisms and interactions that cannot easily be studied on the bulk scale; generally, individual molecular behaviors cannot be distinguished, and only average characteristics can be measured. Nevertheless, the development of the single-molecule sequencer had a signi...
Preprint
Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss...
Preprint
Full-text available
Telomerase reverse transcriptase (TERT) is a protein that catalyzes the reverse transcription of telomere elongation. TERT is also expected to play a noncanonical role beyond telomere lengthening since it localizes not only in the nucleus but also in mitochondria, where telomeres do not exist. Several studies have reported that mitochondrial TERT r...
Article
Full-text available
RNA helicases are enzymes that generally unwind double-stranded RNA using ATP hydrolysis energy, mainly involved in RNA metabolism, transcription, translation, and mRNA splicing. While the helicase core is crucial for RNA unwinding activity, N- and C-terminal extensions of specific helicases may contain an intrinsically disordered region for electr...
Article
Full-text available
Deep-sea Bathymodiolus mussels are generally thought to harbour chemosynthetic symbiotic bacteria in gill epithelial cells called bacteriocytes. However, previously observed openings at the apical surface of bacteriocytes have not been conclusively explained and investigated as to whether the Bathymodiolus symbiosis is intracellular or extracellula...
Article
The author has developed several methodological approaches that use nanophotonic and microfluidic devices to accelerate pharmaceutical research and development. Here, the author describes two of these approaches and provides practical examples. The first is a nanophotonic approach to break the concentration limit of diffusing fluorophore-labeled mo...
Article
A photo-responsive nanovesicle is fabricated by polyion complex (PIC) formation between poly(ethylene glycol) (PEG)-block-polypeptides and photo-reactive oligodeoxynucleotides (PROs)/anti-sense oligonucleotides (ASOs). The ultraviolet (UV) light triggers reversible crosslinking between PROs and ASOs in the vesicular membrane, providing the nanovesi...
Article
Full-text available
SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the a...
Article
Full-text available
In synthetic biology, the control of gene expression requires a multistep processing of biological signals. The key steps are sensing the environment, computing information and outputting products1. To achieve such functions, the laborious, combinational networking of enzymes and substrate-genes is required, and to resolve problems, sophisticated d...
Chapter
Extracellular microRNAs (miRNAs) in body fluids have been identified as promising biomarkers for different human diseases. The high-throughput, multiplexed detection and quantification of these miRNAs are highly beneficial for the rapid and accurate diagnosis of diseases. Here, we developed a simple and convenient microarray-based technique, named...
Article
The Escherichia coli chaperonin GroEL is an essential molecular chaperone that mediates protein folding in association with its cofactor, GroES. It is widely accepted that GroEL alternates the GroES-sealed folding-active rings during the reaction cycle. In other words, an asymmetric GroEL–GroES complex is formed during the cycle, whereas a symmetri...
Article
Full-text available
Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped gene...
Article
Full-text available
The improved catalytic activity of enzymes is required in various fields. Enzymes have conventionally been improved by the screening of bacteria possessing mutant enzymes. However, the screening conditions are limited since screening requires the growth of bacteria. Here, we report the development of a protein microarray for the analysis of enzymat...
Article
Full-text available
SecM, a bacterial secretion monitor protein, contains a specific amino acid sequence at its C-terminus, called arrest sequence, which interacts with the ribosomal tunnel and arrests its own translation. The arrest sequence is sufficient and necessary for stable translation arrest. However, some previous studies have suggested that the nascent chain...
Article
In plasma processing, UV photons generate damage deep in the bulk of transparent materials such as amorphous polymers and glass. In this article, we propose the use of total internal reflection fluorescence microscopy for the nondestructive and highly sensitive detection of UV-induced deep bulk damage and for the first time demonstrate the three-di...
Article
We have developed a protein microwell array for observing enzymatic activity at a density of 1.1×103 spots/cm2. The microarray with 140-μm-diameter microwells was fabricated on a silica glass by dry etching followed by surface modification with Ni-nitrilotriacetic acid (Ni-NTA). β-glucosidase (BGL) tagged with hexa-histidine was synthesized in each...
Conference Paper
We present a novel peptide-based ligand screening system for G protein-coupled receptors (GPCRs) in water-in-oil (W/O) microdroplets. This system is an in vitro gene screening system for peptides that activate a target GPCR expressed in yeast. We show that the system is applicable to human GPCRs. This system will be a powerful tool for identifying...
Conference Paper
We have developed a microfluidic-based fluorescence-activated droplet sorting system using a thermoreversible gelation polymer (TGP) as a switching material. Compared with the conventional fluorescence-activated cell sorter (FACS), TGP-based fluorescence-activated droplet sorting can be performed using a relatively simple optical setup and enables...
Article
Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We have measured the persistence length of DNA molecule for cationic and neutral beta-cyclodextrin binding, using optical tweezers. We propose a methodology for persistence length data analysis based on a quen...
Conference Paper
We present a yeast-based ligand assay system to detect activation of G protein-coupled receptors (GPCRs) in water-in-oil (W/O) droplets. A microfluidic device is used to encapsulate template DNA encoding peptides, a cell-free coupled transcription-translation system, and budding yeast cells expressing GPCR into W/O droplets. The yeast cells are gen...
Article
Full-text available
The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bulle...
Article
Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal comp...
Article
Ethanol produced from cellulosic biomass is generally called 2nd generation bioethanol and is appreciated as an alternative to fossil fuels. In order to make bioethanol, cellulose chains are required to be hydrolyzed into glucose molecules by the cellulases. One of the important enzymes in the process is β-glucosidase (BGL), which catalyzes the hyd...
Article
A single-molecule imaging device using polymeric nanoholes (PNH) was developed. It localizes an excitation volume at the bottom of its nanoholes for the reduction in background noise. Owing to this reduction, the number of types of enzymatic reactions visualized at single-molecule level in PNH was estimated to be approximately 2.5 times greater tha...
Conference Paper
We have developed on-chip sorting systems using sol-gel transition of the thermo-reversible gelation polymer (TGP) as carrier flow controlled by the focused infrared (IR) laser [1]. For efficient sorting, we achieved size and position control of sample core flow in three-dimensional (3D) sheath flow. Stable control was realized under wide range of...
Data
Characterization of chaperonin mutants. (A–C) Protease sensitivity assay. αWT, αWT-His, and αD263C/Q271C/C366S-His (50 nM) were preincubated with or without 1 mM of the different nucleotides (ATP, AMP-PNP, and ADP) for 10 min at 60°C. Digestion with thermolysin (1 ng/µL) was carried out for 10 min at 60°C. The reaction mixtures were precipitated us...
Data
Distributions of time before photobleaching of a single BSR molecule. The surface-immobilized BSR-CPN was observed by epifluorescence microscopy at 23°C and 50°C. The distributions of time before photobleaching of single BSR molecules at 23°C (A) and 50°C (B) were fitted with a single exponential function (solid lines), which yields the rate consta...
Data
Primer sequences used for mutagenesis. (DOC)
Data
The dependence of the fluorescence intensity on temperature. (A) Relative fluorescence intensity of BSR as a function of temperature. The fluorescence intensity at 23°C was taken as 100. The change in fluorescence intensity is well fitted to a single exponential function (solid line). Inset, fluorescence spectra of BSR at 20°C −70°C. (B and C) Dist...
Article
Full-text available
Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding...
Article
The dynamics of full protein synthesis and the co-translational folding processes are not fully understood. We have developed a novel method, using a combination of ribosome display and single-molecule techniques, for monitoring the synthesis, co-translational folding, and maturation of a complete polypeptide chain at the single-molecule level. Thi...
Article
Green fluorescent protein (GFP) has a chromophore that forms autocatalytically within the folded protein. Although many studies have focused on the precise mechanism of chromophore maturation, little is known about the kinetics of de novo chromophore maturation. Here we present a simple and efficient method for examining the de novo kinetics. GFP w...
Article
A novel imaging device with a localized illumination volume beyond the diffraction limit was fabricated by the high-aspect-ratio nanofabrication of a perfluoropolymer. The device had nanoimprinted perfluoropolymer apertures whose diameter and depth were 100 nm and 200 nm, respectively, and was used with a TIRFM. This device is applicable to single-...
Article
Full-text available
It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES)2 complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-Gr...
Article
Controversy exists over whether the chaperonin GroEL forms a GroEL-(GroES)2 complex (football-shaped complex) during its reaction cycle. We have revealed previously the existence of the football-shaped complex in the chaperonin reaction cycle using a FRET (fluorescence resonance energy transfer) assay [Sameshima, Ueno, Iizuka, Ishii, Terada, Okabe...
Article
GroEL is an Escherichia coli chaperonin which is composed of two heptameric rings. GroEL interacts with its cofactor GroES and assists protein folding in an ATP dependent manner. Because of negative cooperativity between two rings of GroEL in the binding of ATP, it has been generally believed that an asymmetrical 1:1 complex is only a functional fo...
Article
Bacterial ribosome is a molecular machine composed of 30S and 50S subunits that translates the genetic code in mRNA into an amino acid sequence through repetitive cycles of tRNA selection, peptide bond formation and translocation. Translation initiation is one of the essential processes in protein synthesis that involves the assembly of initiator f...
Article
A single-molecule fluorescence imaging device was developed using the amorphous perfluoropolymer Cytop™ (Asahi Glass Co., Ltd.). It was fabricated by imprint lithography, plasma etching and plasma hydrophilization, which we developed for the perfluoropolymer. In total internal reflection fluorescence microscopy (TIRFM), the device confines the exci...
Article
Prefoldin (PFD) is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin (CPN) for correct folding. However, mechanism of substrate transfer from PFD to CPN remains to be elucidated. Previous studies showed that CPN has a helical protrusion as a built-in-lid, and uses ATPase cycling to promote...
Article
There exist two small heat shock proteins (sHsps) in the fission yeast, Schizosaccharomyces pombe (S. pombe), whose expressions are highly induced by heat stress. We have previously expressed, purified, and characterized one of the sHsps, SpHsp16.0. In this study, we examined the other sHsp, SpHsp15.8. It suppressed the thermal aggregation of citra...
Article
Full-text available
ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformatio...
Article
Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of The...
Article
To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by ∼2 s in comparison with that of the wild type, without interf...
Article
Full-text available
How folding of proteins is coupled to their synthesis remains poorly understood. Here, we apply single-molecule fluorescence imaging to full protein synthesis in vitro. Ribosomes were specifically immobilized onto glass surfaces and synthesis of green fluorescent protein (GFP) was achieved using modified commercial Protein Synthesis using Recombina...
Article
Full-text available
To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by ∼2 s in comparison with that of the wild type, without interf...