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GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES,(More)
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 Recombinant(More)
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 bullet-shaped(More)
Prefoldin is a chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. However, kinetics of interactions between prefoldin and substrate proteins have not been investigated. In this study, dissociation constants and dissociation rate constants of unfolded proteins with prefoldin were firstly(More)
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 approximately 2 s in comparison with that of the wild type, without(More)
Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and(More)
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 and(More)
We report a novel method of one-step direct amination on polycrystalline diamond to produce functionalized surfaces for DNA micropatterning by photolithography. Polycrystalline diamond was exposed to UV irradiation in ammonia gas to generate amine groups directly. After patterning, optical microscopy confirmed that micropatterns covered with an Au mask were(More)
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-GroES(More)
A simple and efficient technique is described for measuring photoluminescence (PL) maps of carbon nanotubes (NTs) in the extended IR range (1-2.3 mum). It consists of preparing an NT/surfactant/gelatin film and measuring PL spectra using a combination of a tunable Ti-sapphire laser excitation and FTIR detection. This procedure has been applied to a wide(More)