Nucleotide and Mg2+ induced conformational changes in GroEL can be detected by sulfhydryl labeling

The accessibility of fluorescein-5-maleimide to sulfhydryl groups in the mo lecular chaperone GroEL was used to follow structural rearrangements in the protein triggered by binding Mg2+ and/or adenine nucleotides. Three peptid es, each containing one of the cysteines of GroEL (C138, C458 and C519) wer e identified. GroEL labeled in 50mM TrisHCl, pH 7.8, incorporated similar t o 0.3 labels each on C138 and C458. With 10mM MgCl2, the labeling increased to similar to 0.8 labels each on C138 and C458. The increase was partially due to a conformational change which occurred upon Mg2+ binding as well as to an increase in ionic strength. When ADP, ATP, or AMP-PNP were added to a solution of GroEL and Mg2+, C138 incorporated similar to 0.8 labels, whil e C458 incorporated similar to 0.1 labels. These results suggest that the b inding of adenine nucleotides changed the conformation of GroEL and made a previously highly exposed sulfhydryl group inaccessible. GroEL slowly disso ciated into monomers when it was extensively labeled at C458. GroEL labeled with fluorescein-5-maleimide, under any of the conditions examined, was ab le to bind but not release active rhodanese. The observed variations in sul fhydryl accessibility are consistent with mechanisms that suggest binding o f GroES to GroEL differs from the binding of substrate protein to GroEL, an d that the binding of Mg2+ or Mg-adenine nucleotides results in conformatio nal changes in GroEL.