Abstract
Two heptamer rings of chaperonin GroEL undergo opening-closing conformational transition in the reaction cycle with the aid of GroES and ATP. We introduced Cys into the GroEL subunit at Ala-384 and Ser-509, which are very close between adjacent GroEL subunits in the open heptamer ring but far apart in the closed heptamer ring. The open ring-specific inter-subunit cross-linking between these Cys indicated that the number of rings in open conformation in GroEL was two in ATP (GroEL(OO)), one in ADP (GroEL(O)), and none in the absence of nucleotide. ADP showed an inhibitory effect on ATP-induced generation of GroEL(OO). The isolated GroEL(O) and GroEL(OO), which lost any bound nucleotide, could bind GroES to form a bullet-shaped 1:1 GroEL-GroES complex and a football-shaped 1:2 GroEL-GroES complex, respectively, even without the addition of any nucleotide. Substrate protein was unable to form a stable complex with GroEL(OO) and did not stimulate ATPase activity of GroEL. These results favor a model of the GroEL reaction cycle that includes a football complex as a critical intermediate.
Highlights
One of two heptamer rings in GroEL (14 –16)
We report here the opposite version; open conformation-specific intersubunit cross-links were introduced into the GroEL ring. Using this cross-linking as a probe of open conformation, we found that one ring was open in ADP (GroELO), two rings were open in ATP (GroELOO)
Specific Cross-linking for the Open Ring Was Designed—In the crystal structure of 1:1 GroEL-GroES bullet complex containing ADP (Protein Data Bank code 1AON [14]), Ala-384 of one GroEL subunit in the open heptamer ring attached by a GroES lid is very close (C␣-C␣ distance, ϳ6.4 Å) to Ser-509 of the neighboring subunit (Fig. 1)
Summary
Reagents and Proteins—Pig heart mitochondrial malate dehydrogenase (MDH) and NADH were purchased from Roche Applied Science; ATP, ADP, pyruvate kinase, lactate dehydrogenase, and hexokinase were from Sigma. The trace amount of contaminating ATP in the ADP solution was eliminated by hexokinase/glucose treatment [24]. Mutant proteins were generated by site-directed mutagenesis using the PrimeSTAR mutagenesis basal kit from Takara. GroEL mutants and GroES were purified as described previously [25, 26]. Dithiothreitol was removed from the stored GroEL by ammonium sulfate precipitation. The stored GroEL was precipitated by 65% saturated ammonium sulfate, and precipitation was washed by HKM buffer containing 65% saturated ammonium sulfate and resolved in HKM buffer. Cy3-labeled GroES (GroES-Cy3) and BODIPYlabeled MDH (MDH-BODIPY) were prepared as described previously [28, 29].
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