A reversible conformational transition in muscle actin is caused by nucleotide exchange and uncovers cysteine in position 10.

ATP-G-actin in the absence of excess ATP and divalent metal ions was treated with ADP in amounts large enough to ensure complete formation of ADP-G-actin. Under these conditions the monomer undergoes a very slow structural transition as seen by the exposure of 2.0 +/- 0.2 thiol groups per actin molecule. Once exposed, the second thiol group reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) at a rate approximately 10-fold higher than that of cysteine 374. Labeling experiments with 2,4-dinitrophenyl [1-14C]cysteinyl disulfide followed by digestion and peptide analysis showed (besides reaction with cysteine 374) nearly exclusive labeling of cysteine 10. Since this residue is completely shielded in ATP-G-actin, exchange of ATP for ADP must have caused a partial unfolding of the protein uncovering the side chain of this cysteine. The transition is reversible, because addition of ATP or of excess divalent metal ions restored the conformation with only cysteine 374 exposed. Reversibility of the transition allowed us to directly determine the relative affinities of ATP and ADP to monomeric actin in the absence of Me2+ ions. By determination of the 50% exposure value of cysteine 10 from either side of the equilibrium we found a value of KATP/KADP = 30. The rate of uncovering of the thiol of cysteine 10 at 0 degree C was distinctly slower (t1/2 = 9 h) than its reshielding by the addition of ATP (t1/2 = 3 h). The structural change was accompanied by a decrease in polymerization rate. Relative polymerization rates were determined as ATP-G(1S)-actin:ADP-G(approximately 1S)-actin:ADP-G(2S)-actin = 1.0:0.35:0.1. From the data presented here we conclude that preparations of ADP-G-actin remain undefined unless the number of thiol groups exposed has been determined.