Single-Molecule Imaging of 1:2 Groel-Groes Complexes in Zero-Mode Waveguides

Abstract

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 form for over a decade. Contrary to the belief, we revealed that a symmetrical 1:2 GroEL-GroES complex can be formed in the presence of denatured protein using fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). However, the dynamics of GroEL-GroES interaction including a 1:2 GroEL-GroES complex is still unclear. To clarify this issue, 1:2 GroEL-GroES complexes were observed using zero-mode waveguides (ZMWs) at a single molecule level. ZMWs are nanoholes array fabricated in a thin metal film. They can reduce excitation volume compared to total internal reflection illumination; therefore, they make it possible to observe individual 1:2 GroEL-GroES complexes at sub-μM concentrations that are required to form the complexes. Cy3-GroES and Cy5-GroEL binding to and dissociating from Alexa488-GroES immobilized on the bottoms of ZMWs were visualized. Cy3-GroES and Cy5-GroEL were co-localized in GroES-immobilized ZMWs for ∼ 3 s. The duration time in ZMWs without immobilizing Alexa488-GroES, which reflected non-specific adsorption of GroEL-GroES complex to the bottoms of ZMWs, was ∼ 1 s. These results showed that 1:2 GroEL-GroES complexes were successfully observed at a single-molecule level and their duration time was estimated to be ∼3 s. Furthermore, the same experiment was carried out in the absence of denatured protein. The duration time of Cy3-GroES and Cy5-GroEL in Alexa488-GroES-immobilized ZMWs was ∼1 s. This result indicated that 1:2 GroEL-GroES complexes disappeared in the absence of denatured protein, being consistent with FRET and FCS experiments.