Topology of the morphological domains of the chaperonin GroEL visualized by immuno-electron microscopy.

Abstract

Electron microscopy of the tetradecameric double-ring complex of GroEL reveals a four-layered structure, indicating that the 58 kDa subunits are composed of two major morphological domains. We have used immuno-electron microscopy to assign these domains to the corresponding segments of the GroEL sequence. Upon chemical modification of GroEL with N-ethylmaleimide, protease treatment in the presence of ATP or ADP generates GroEL fragments of 15 kDa (N15; residues 1-141) and 40 kDa (C40; residues 153-531). As visualized by scanning transmission electron microscopy, affinity-purified antibodies directed against C40 recognize the outer layers, whereas antibodies against N15 interact with the equatorial portions of the GroEL double-ring. Thus, the two major domains of the subunits in the chaperonin complex are arranged in the order C40-N15:N15-C40. The single-ring chaperonin co-factor GroES interacts with the C40 domain while the ATP-binding site of GroEL is probably close to the junction between N15 and C40.