Fluorescence detection of symmetric GroEL₁₄(GroES₇)₂ heterooligomers involved in protein release during the chaperonin cycle

The GroEL₁₄ chaperonin from Escherichia coli was labeled with 5- ((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (I-AEDANS), a hydrophobic probe whose fluorescent emission is sensitive to structural changes within the protein. Increasing concentrations of ATP or adenylyl imidodiphosphate but not ADP caused two successive GroES₇-dependent changes in the fluorescence intensity of AEDANS-GroEL₁₄, corresponding to the sequential binding of two GroES₇ heptamers and the formation of two types of chaperonin heterooligomers, GroEL₁₄GroES₇ and GroEL₁₄(GroES₇)₂. The binding of thermally denatured malate dehydrogenase (MDH) caused a specific increase in fluorescence intensity of AEDANS-GroEL₁₄ that allowed the direct measurement in solution at equilibrium of ATP- and GroES₇-dependent protein release from the chaperonin. Structure/function analysis during the generation of ATP from ADP indicated the following sequence of events: 1) ADP-stabilized MDH-GroEL₁₄GroES₇ particles bind newly formed ATP. 2) MDH- GroEL₁₄GroES₇ particles bind a second GroES₇. 3) MDH-GroEL₁₄(GroES₇)₂ particles productively release MDH. 4) Released MDH completes folding. Therefore, the symmetrical GroEL₁₄(GroES₇)₂ heterooligomer is an intermediate after the formation of which the protein substrate is productively released during the chaperonin-mediated protein folding cycle.