Metal Nanoparticles Dispersion By Chaperonin Complexes
Authors: Yoda Hiromi , Koike-Takeshita Ayumi 
Published: February 2020

Introduction
GroEL is the best characterized chaperonin; it is found in the cytoplasm of Escherichia coli and is essential for cell viability and growth. GroEL is a large cylindrical protein complex comprising two heptameric rings of identical 57-kDa subunits, and these rings are stacked back to back. GroES is a single heptameric ring of identical 10-kDa subunits. GroEL encapsulates the substrates to the cavities, GroEL binds a wide variety of substrate proteins in non-native states, and forms a binary complex, which then binds seven ATP molecules with positive cooperativity and GroES to the same (cis) GroEL ring, to form the cis-ternary complex. The binding of GroES induces the encapsulation of the substrate protein into an enlarged cavity (horizonal width of 5-6nm and vertical width of 6-7nm) inside the cis-ring, which is capped by GroES. In the cis-cavity, non-native protein initiates folding without the risk of aggregation. Following hydrolysis of ATP in the cisring, the products of this cycle, ADP and folded substrate protein, are released with GroES, as another similar folding-active assembly is formed in the opposite trans ring. On the other hand, the hydrolysis-defective mutant GroEL(D52/398A) can bind two GroES in the presence of ATP, producing a symmetric 1:2 GroEL-GroES complex (the “football”-shaped complex) with half-time of ~150 h (~6 days). The property of the GroEL(D52/398A) looks beneficial for dispersing aggregative nanoparticles and hydrophobic compounds. FePt, Pt, CdSe/ZnS and Au were used to encapsulate in GroEL(D52/398A) as metal nanoparicles because these nanoparticles are used to engineering manufactures such as semiconductors, devices and batteries.
Experiments
Pt (average φ2 nm), FePt (φ4 nm), CdSe/ZnS (φ7nm and 6 nm) and Au (φ10 nm, 4 nm and 2 nm) were used to encapsulate in GroEL(D52/398A) as metal nanoparicles. In the case of nanoparticle encapsulation in one side of GroEL(D52/398A) cavities, 0.1-1.0 µM GroEL(D52/398A), 0.2-2.0 µM GroES (2-fold mol of GroEL), and 1 mM ATP were added to the sonicated nanoparticle solution. For two different nanoparticle encapsulation in both sides of GroEL(D52/398A) cavities, GroEL(D52/398A) which is bound to first nanoparticle was added to the same molar of GroES and 5 mM ADP. The bullet-shaped GroEL(D52/398A) encapsulating first nanoparticle was mixed with the second nanoparticle solution for 1 minute, then, GroES and 1 mM ATP were added to form the football-shaped complex. GroEL(D52/398A) complexes were stained with 1% phosphotungstic acid (pH 4.0) for the observation with the transmission electron microscopy JEM 2000EX or JEM 2100 (JEOL Co., Ltd.). For protein crystalizaion, the sample solution of metal nanoparticle encapsulating GroEL(D52/398A) complexes was added to 1% ammonium molybdate/0.2 % PEG1500 with equal amount volume. Then, the mixture was dropped on TEM grid or flesh cleaved mica and air dried. Fixed protein was observed using with TEM or scanning probe microscopy SPM 9600 (Shimazu Corp.). Analysis was performed using with gel filtration HPLC system equipped with a G3000SWXL (TOSOH) and elution buffer which was consisted of 20 mM HEPES/KOH (pH 7.0), 100 mM KCl, 5 mM MgCl2 and 50 mM Na2SO4 at a flow rate of 0.5 ml/min. The eluate was monitored using a UV detector set at the wavelength 280 nm. Fluorescence intensity was detected at an excitation wavelength of 370 nm and emission at 490 nm for CdSe/ZnS λ490 or 575 nm emission wavelength for CdSe/ZnS λ575 was used.
Results and discussion
From TEM images, the highest FePt nanoparticles encapsulation percentage of GroEL(D52/398A) was about 95% in this experimental condition. Pt, CdSe/ZnS and Au nanoparticles were encapsulated in GroEL(D52/398A) complexes, too. GroEL(D52/398A) wasn’t seemed to select metal nanoparticle. Two CdSe/ZnS nanoparticles encapsulation of GroEL(D52/398A) of analysis indicated FL emission peaks of both wave length at 490 nm and 575 nm around 12 min of retention time which GroEL(D52/398A) elution time with UV detector. This result showed a possibly that GroEL(D52/398A) could encapsulate two particles in each their cavities. In a presence of protein crystallization reagent, metal nanoparticles encapsulating GroEL(D52/398A) complexes showed tendency to fill up with themselves on TEM grid or mica as an experimental flat plane. These experiments are continued to make monolayer aligns of metal nanoparticle encapsulating GroEL(D52/398A) complexes on flat plated of manufactures. These results from a sequential experiments of GroEL(D52/398A) suggest a potential that GroEL(D52/398A) will support to construct nano manufactures.